Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same

ABSTRACT

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses human immunoglobulin light chain variable domains derived from a limited repertoire of human immunoglobulin light chain variable gene segments that comprise histidine modifications in their germline sequence. Methods of making non-human animals that express antibodies comprising histidine residues encoded by histidine codons introduced into immunoglobulin light chain nucleotide sequences are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. patent application Ser. No.14/030,424, filed Sep. 18, 2013, which is a continuation-in-part of U.S.patent application Ser. No. 13/832,247, filed Mar. 15, 2013, whichclaims benefit under 35 U.S.C. §119(e) of U.S. Provisional ApplicationNo. 61/611,950, filed Mar. 16, 2012, and U.S. Provisional ApplicationNo. 61/736,930, filed Dec. 13, 2012, all incorporated by referenceherein in their entireties.

FIELD OF INVENTION

A genetically modified non-human animal (e.g., rodent, e.g., mouse orrat) is provided that expresses antibodies capable of binding to anantigen in a pH dependent manner. A method for making modifications toimmunoglobulin light chain variable region sequence of a non-humananimal is provided, wherein the modifications include the mutagenesis ofresidues within the light chain variable region gene, e.g., nucleotidesthat encode one or more amino acids within a complementary determiningregion (CDR), to facilitate in vivo expression of antibodies comprisinglight chain domains that exhibit pH dependent binding to antigens.Methods for making antibodies with pH-dependent antigen binding are alsoprovided.

BACKGROUND OF THE INVENTION

Antibodies typically comprise a homodimeric heavy chain component,wherein each heavy chain monomer is associated with an identical lightchain. Antibodies having a heterodimeric heavy chain component (e.g.,bispecific antibodies) are desirable as therapeutic antibodies. Butmaking bispecific antibodies having a suitable light chain componentthat can satisfactorily associate with each of the heavy chains of abispecific antibody has proved problematic.

In one approach, a light chain might be selected by surveying usagestatistics for all light chain variable domains, identifying the mostfrequently employed light chain in human antibodies, and pairing thatlight chain in vitro with the two heavy chains of differing specificity.

In another approach, a light chain might be selected by observing lightchain sequences in a phage display library (e.g., a phage displaylibrary comprising human light chain variable region sequences, e.g., ahuman scFv library) and selecting the most commonly used light chainvariable region from the library. The light chain can then be tested onthe two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phagedisplay library of light chain variable sequences using the heavy chainvariable sequences of both heavy chains of interest as probes. A lightchain that associates with both heavy chain variable sequences might beselected as a light chain for the heavy chains.

In another approach, a candidate light chain might be aligned with theheavy chains' cognate light chains, and modifications are made in thelight chain to more closely match sequence characteristics common to thecognate light chains of both heavy chains. If the chances ofimmunogenicity need to be minimized, the modifications preferably resultin sequences that are present in known human light chain sequences, suchthat proteolytic processing is unlikely to generate a T cell epitopebased on parameters and methods known in the art for assessing thelikelihood of immunogenicity (i.e., in silico as well as wet assays).

All of the above approaches rely on in vitro methods that subsume anumber of a priori restraints, e.g., sequence identity, ability toassociate with specific pre-selected heavy chains, etc. There is a needin the art for compositions and methods that do not rely on manipulatingin vitro conditions, but that instead employ more biologically sensibleapproaches to making human epitope-binding proteins that include acommon light chain.

In addition, therapeutic antibodies, e.g., bispecific therapeuticantibodies, have some limitations in that they often require high dosesto achieve desired efficacy. This is partly due to the fact thatantibody-antigen complexes are internalized into the endosome, and aretargeted for lysosomal degradation in a process called target-mediatedclearance. Thus, there is a need in the art for methods and compositionsthat lead to more efficient antibody recycling, e.g., bispecificantibody recycling, and prevent degradation of the antibody by promotingdissociation of antibody-antigen complexes in the endosomal compartmentwithout compromising the specificity and affinity of the antibody towardthe antigen.

SUMMARY OF THE INVENTION

In one aspect, a biological system is provided for generating anantibody or an antibody variable domain that binds a target antigen at aneutral pH but exhibits reduced binding of the same antigen at an acidicpH (e.g., pH 5.0-6.0). The biological system comprises a non-humananimal, e.g., a rodent (e.g, a mouse or rat) that has a rearranged lightchain sequence (e.g., a rearranged V-J) that comprises one or morehistidine modifications. In various aspects, the one or more histidinemodifications are in the light chain CDR3 codon. In various aspects, thenon-human animal comprises a human or humanized heavy chainimmunoglobulin locus. In various aspects, the non-human animal comprisesa replacement of endogenous non-human heavy chain variable gene segmentswith one or more human heavy chain V_(H), D_(H), and J_(H) segments,wherein the human segments are operably linked to a non-humanimmunoglobulin constant region. In various aspects, non-human animalswith universal light chains comprising light chain variable domains withsubstitutions of non-histidine residues for histidine residues areprovided. In various aspects these histidine-modified universal lightchain non-human animals (e.g., rodents, e.g., mice) are referred to ashistidine-universal light chain mice, histidine-ULC mice, or HULC mice.

Thus, in one aspect, provided herein is a genetically modified non-humananimal that comprises in its germline an immunoglobulin light chainlocus that comprises a single rearranged human immunoglobulin lightchain variable region gene sequence comprising human V_(L) and J_(L)segment sequences, wherein the single rearranged human immunoglobulinlight chain variable region sequence comprises a substitution of atleast one non-histidine codon with a histidine codon. In one embodiment,the single rearranged human immunoglobulin variable region sequence isoperably linked to an immunoglobulin light chain constant region genesequence. In one embodiment, the immunoglobulin light chain constantregion gene sequence is a non-human immunoglobulin light chain constantregion gene sequence. In one embodiment, the non-human immunoglobulinlight chain constant region gene sequence is an endogenousimmunoglobulin light chain constant region gene sequence. In oneembodiment, the non-human animal lacks a functional unrearrangedimmunoglobulin light chain variable region. In one embodiment, theimmunoglobulin light chain locus is at an endogenous non-humanimmunoglobulin light chain locus.

In one embodiment, the animal further comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region gene sequence comprisinghuman V_(H), D_(H), and J_(H) segments operably linked to animmunoglobulin heavy chain constant region gene sequence. In oneembodiment, the immunoglobulin heavy chain constant region gene sequenceis a non-human heavy chain constant region gene sequence. In oneembodiment, the non-human heavy chain constant region gene sequence isan endogenous immunoglobulin heavy chain constant region gene sequence.In one embodiment, the immunoglobulin heavy chain locus is at anendogenous immunoglobulin heavy chain locus.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR). In one embodiment, thesubstitution of at least one non-histidine codon with a histidine codonis in the nucleotide sequence encoding a CDR3. In one embodiment, thesubstitution is of one, two, three, four, or more CDR3 codons. In oneaspect, the single rearranged human immunoglobulin light chain variableregion sequence comprised at the immunoglobulin light chain locus isderived from a human Vκ1-39 or Vκ3-20 gene segment. In one embodiment,the Vκ sequence of the human Vκ1-39 or Vκ3-20 gene segment is a germlineVκ1-39 or Vκ3-20 sequence but for the histidine modifications. In oneembodiment, the single rearranged human immunoglobulin light chainvariable region is derived from a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1gene sequence. In one embodiment, the single rearranged humanimmunoglobulin light chain variable region is derived from a rearrangedVκ1-39/Jκ5 gene sequence, and the Vκ1-39/Jκ5 gene sequence comprises areplacement of at least one non-histidine codon with a histidine codondesigned to express a histidine at a position selected from 105, 106,108, 111, and a combination thereof. In another embodiment, the singlerearranged human immunoglobulin light chain variable region is derivedfrom a rearranged Vκ3-20/Jκ1 gene sequence, and the Vκ3-20/Jκ1 genesequence comprises a replacement of at least one non-histidine codonwith a histidine codon designed to express a histidine at a positionselected from 105, 106, 107, 109, and a combination thereof.

In one aspect, the non-human animal described herein comprises apopulation of B cells in response to an antigen of interest that isenriched for antibodies that exhibit a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, the decrease in t_(1/2) at an acidic pH as comparedto a neutral pH is about 30 fold or more. In one embodiment, suchenrichment is at least about 2 fold.

In one embodiment, the animal expresses an antibody comprising a humanimmunoglobulin light chain variable domain with a substitution of atleast one non-histidine residue with a histidine residue at an aminoacid position encoded by the at least one codon substituted in theimmunoglobulin light chain variable region gene sequence. In oneembodiment, the animal expresses an antibody that retains a substitutionof at least one non-histidine residue with a histidine residue in anexpressed human immunoglobulin light chain variable domain, despitesomatic hypermutations.

In one embodiment, the non-human animal is a mammal. In one embodiment,the mammal is a rodent, e.g., a rat or a mouse. In one embodiment, thenon-human animal is a mouse. Thus, in one aspect, also provided hereinis a genetically modified mouse comprising in its germline animmunoglobulin light chain locus that comprises a single rearrangedhuman immunoglobulin light chain variable region gene sequencecomprising human V_(L) and J_(L) segment sequences, wherein the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon. In one embodiment, the mouse lacks a functionalunrearranged immunoglobulin light chain variable region.

In one embodiment, the single rearranged immunoglobulin light chainvariable region gene sequence in the germline of the mouse is operablylinked to an immunoglobulin light chain constant region gene sequence.In one embodiment, the immunoglobulin light chain constant region genesequence is selected from a rat or a mouse immunoglobulin light chainconstant region gene sequence. In one embodiment, the immunoglobulinlight chain constant region gene sequence is a mouse sequence. In oneembodiment, the immunoglobulin light chain locus is at an endogenousmouse immunoglobulin light chain locus.

In a further embodiment, the mouse also comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region sequence comprising humanV_(H), D_(H), and J_(H) segments operably linked to an immunoglobulinheavy chain constant region gene sequence. In one aspect, theimmunoglobulin heavy chain constant region gene sequence is a rat or amouse heavy chain constant region gene sequence. In one embodiment, theimmunoglobulin heavy chain constant region gene sequence is a mousesequence. In one embodiment, the immunoglobulin heavy chain locus is atan endogenous mouse immunoglobulin heavy chain locus.

In one aspect, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon wherein the substitution isin the nucleotide sequence encoding a CDR. In one embodiment, thesubstitution is in a CDR3 codon, e.g., in one, two, three, four, or moreCDR3 codons. In one embodiment, the immunoglobulin light chain locus ofthe mouse comprises the single rearranged human immunoglobulin lightchain variable region sequence derived from a human Vκ1-39 or Vκ3-20gene segment, e.g., the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, the single rearrangedimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5 gene sequence and the Vκ1-39/Jκ5 sequencecomprises a replacement of at least one non-histidine codon with ahistidine codon designed to express a histidine at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In one embodiment,such replacement is designed to replace histidines at positions 105,106, 108, and 111. In another embodiment, such replacement is designedto replace histidines at positions 106, 108, and 111.

In another embodiment, the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ3-20/Jκ1 genesequence and the Vκ3-20/Jκ1 sequence comprises a replacement of at leastone non-histidine codon with a histidine codon designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, such replacement is designed toreplace histidines at positions 105, 106, 107, and 109. In anotherembodiment, such replacement is designed to replace histidines atpositions 105, 106, and 109.

In one embodiment, the mouse described herein comprises a population ofB cells in response to an antigen of interest that is enriched forantibodies that exhibit a decrease in dissociative half-life (t_(1/2))at an acidic pH as compared to neutral pH of at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 25-fold, or at least about 30-fold. In one embodiment, thedecrease in t_(1/2) at an acidic pH as compared to a neutral pH is about30 fold or more. In one embodiment, such enrichment of antibodies is atleast about 2 fold.

In one embodiment, the mouse described herein expresses a population ofantigen-specific antibodies in response to an antigen of interestwherein all antibodies comprise (a) immunoglobulin light chain variabledomains derived from the same single rearranged human light chainvariable region gene sequence which comprises a substitution of at leastone non-histidine codon with a histidine codon, and (b) immunoglobulinheavy chains comprising heavy chain variable domains derived from arepertoire of human heavy chain V, D, and J segments.

Also provided herein is a non-human locus, e.g., mouse locus, comprisinga single rearranged human immunoglobulin light chain variable regiongene sequence comprising human V_(L) and J_(L) segment sequences,wherein the single rearranged human immunoglobulin light chain variableregion gene sequence comprises a substitution of at least onenon-histidine codon with a histidine codon. In one embodiment, the locusis comprised in the germline of a non-human animal. In one embodiment,the locus comprises the single rearranged human immunoglobulin lightchain variable region gene sequence derived from a human Vκ1-39 orVκ3-20 gene segment, e.g., derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, wherein the singlerearranged human immunoglobulin light chain variable region genesequence present in the locus is derived from the rearranged Vκ1-39/Jκ5sequence, the substitution of at least one non-histidine codon with ahistidine codon is designed to express a histidine at a positionselected from 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the single rearranged human immunoglobulin lightchain variable region gene sequence present in the locus is derived fromthe rearranged Vκ3-20/Jκ1 sequence, the substitution of at least onenon-histidine codon with a histidine codon is designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In various embodiments, the non-human locidescribed herein may be generated using methods described below formaking a genetically modified non-human animal.

In yet another aspect, provided herein is a method for making anon-human animal that comprises a genetically modified immunoglobulinlight chain locus in its germline, wherein the method comprisesmodifying a genome of a non-human animal to delete or rendernon-functional endogenous immunoglobulin light chain V and J segments inan immunoglobulin light chain locus, and placing in the genome a singlerearranged human light chain variable region gene sequence comprising asubstitution of at least one non-histidine codon with a histidine codon.In one embodiment, such method results in a genetically modifiednon-human animal that comprises a population of B cells enriched forantibodies exhibiting pH-dependent binding to the antigen of interest.In one embodiment, the single rearranged human immunoglobulin lightchain variable region sequence placed in the genome is derived from ahuman Vκ1-39 or Vκ3-20, e.g., a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 genesequence. Thus, in the embodiment wherein the single rearranged humanimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5, the substitution of at least one non-histidinecodon with a histidine codon is designed to express a histidine at aposition selected from 105, 106, 108, 111, and a combination thereof. Inan embodiment wherein the single rearranged human immunoglobulin lightchain variable region sequence is derived from a rearranged Vκ3-20/Jκ1,the substitution of at least one non-histidine codon with a histidinecodon is designed to express a histidine at a position selected from105, 106, 107, 109, and a combination thereof.

In another aspect, provided herein is a method of generating an antibodythat exhibits pH-dependent binding to an antigen of interest comprising(a) generating a mouse described herein (e.g., a mouse that comprises inits germline an immunoglobulin light chain locus that comprises a singlerearranged human immunoglobulin light chain variable region sequencecomprising human V_(L) and J_(L) segment sequences and a substitution ofat least one non-histidine codon with a histidine codon in itsrearranged light chain variable region sequence), (b) immunizing themouse with an antigen of interest, and (c) selecting an antibody thatbinds to the antigen of interest with a desired affinity at a neutral pHwhile displaying reduced binding to the antigen at an acidic pH. In oneembodiment, the method results in a generation of an antibody thatexhibits t_(1/2) at acidic pH and 37° C. of about 2 minutes or less. Inone embodiment, the method results in a generation of an antibody thatdisplays a decrease in dissociative half-life (t_(1/2)) at an acidic pHas compared to neutral pH of at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about25-fold, or at least about 30-fold.

In other aspects, provided herein are additional methods of generatingan antibody that exhibits pH-dependent binding to an antigen ofinterest. One such method comprises (a) selecting a first antibody thatbinds to an antigen of interest with a desired affinity, (b) modifyingan immunoglobulin light chain nucleotide sequence of the first antibodyto comprise a substitution of at least one non-histidine codon with ahistidine codon, (c) expressing an immunoglobulin heavy chain of thefirst antibody and the modified immunoglobulin light chain in a cell,and (d) selecting a second antibody expressed in the cell that retains adesired affinity for the antigen of interest at neutral pH and displaysreduced binding to the antigen of interest at an acidic pH. In oneembodiment, the immunoglobulin light chain nucleotide sequence of thefirst antibody comprises a single rearranged human immunoglobulin lightchain variable region sequence. In one embodiment, the first antibody isgenerated in a non-human animal, e.g., a mouse, comprising animmunoglobulin light chain sequence derived from a single rearrangedhuman immunoglobulin light chain variable region sequence, and themodification of the immunoglobulin light chain is made in the singlerearranged human immunoglobulin variable region sequence. In oneembodiment, the first antibody is generated in a non-human animal, e.g.,a mouse, further comprising an immunoglobulin heavy chain sequencederived from a repertoire of human V_(H), D_(H), and J_(H) segments. Inone embodiment, the single rearranged human immunoglobulin light chainvariable region sequence is selected from Vκ1-39/Jκ5 and Vκ3-20/Jκ1 genesequence. In an embodiment, wherein the single rearranged humanimmunoglobulin light chain variable region sequence is Vκ1-39/Jκ5, themodification in the immunoglobulin light chain nucleotide sequence ofthe first antibody is made in the CDR3 codon at a position selected from105, 106, 108, 111, and a combination thereof. In an embodiment whereinthe single rearranged human immunoglobulin light chain variable regionsequence is Vκ3-20/Jκ1, the modification in the immunoglobulin lightchain nucleotide sequence of the first antibody is made in the CDR3codon at a position selected from 105, 106, 107, 109, and a combinationthereof.

In one embodiment, the method of generating an antibody that exhibitspH-dependent binding to an antigen of interest described herein resultsin an antibody that displays a decrease in dissociative half-life(t_(1/2)) at an acidic pH as compared to neutral pH of at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold. In oneembodiment, the method of generating the antibody results in an antibodythat exhibits a t_(1/2) at acidic pH and 37° C. of about 2 minutes orless.

In other various aspects, provided herein is a genetically modifiednon-human animal, e.g., a mouse, that comprises a limited repertoire oflight chain variable gene segments, e.g., no more than two human V_(L)gene segments and one or more, e.g., two or more, human J_(L) genesegments operably linked to a mouse or rat light chain constant region,and one or more human V_(H), one or more human D_(H), and one or morehuman J_(H) gene segments, operably linked to a non-human constantregion; wherein the human gene segments are capable or rearranging andencoding human variable domains of an antibody, and further wherein themouse does not comprise an endogenous V_(L) gene segment that is capableof rearranging to form an immunoglobulin light chain. In one embodiment,the light chain constant region is a rat or a mouse constant region,e.g., a rat or a mouse Cκ constant region. In one embodiment, the mousecomprises five human Jκ gene segments, e.g., Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5gene segments. In one embodiment, the no more than two human V_(L) genesegments are selected from a human Vκ1-39, Vκ3-20, and a combinationthereof, e.g., the two human V_(L) gene segments are Vκ1-39 and Vκ3-20.In one embodiment, the no more than two human V_(L) gene segments andone or more, e.g., two or more, human J_(L) gene segments are present atthe endogenous light chain locus, e.g., endogenous kappa light chainlocus. In one embodiment, the mouse comprises a functional λ, lightchain locus. In another embodiment, the mouse comprises a non-functionalλ, light chain locus. In one embodiment, the one or more human V_(H),one or more human D_(H), and one or more human J_(H) gene segments areoperably linked to a mouse or a rat heavy chain constant regionsequence.

In some embodiments, also provided herein is a non-human locuscomprising a limited repertoire of human variable gene segments, e.g., anon-human locus comprising no more than two human V_(L) gene segmentsand one or more, e.g., two or more, human J_(L) gene segments operablylinked to an immunoglobulin constant region sequence (e.g., a non-humanimmunoglobulin constant region sequence, e.g., a rat or a mousesequence). In one embodiment, the locus comprises five human Jκ genesegments, e.g., Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 gene segments. In oneembodiment, the no more than two human V_(L) gene segments are selectedfrom Vκ1-39 and Vκ3-20, and a combination thereof (e.g., no more thantwo human V_(L) gene segments are Vκ1-39 and Vκ3-20). In variousembodiments, the non-human loci described herein may be generated usingmethods described throughout this application for making geneticallymodified non-human animals. Thus, a method of making a geneticallymodified non-human animal comprising a limited repertoire of humanvariable gene segments, e.g., comprising no more than two human V_(L)gene segments and one ore more, e.g., two or more, human J_(L) genesegments operably linked to an immunoglobulin constant region sequence(e.g., a non-human immunoglobulin constant region sequence, e.g., a rator a mouse sequence) is also provided.

In various aspects, a mouse is provided that expresses an immunoglobulinlight chain generated from a rearrangement of one of two human Vκ genesegments and one of 1, 2, 3, 4, or 5 human Jκ gene segments, wherein themouse comprises a replacement of all or substantially all endogenousimmunoglobulin V_(H) gene segments with one or more human immunoglobulinV_(H), one or more D_(H), and one or more J_(H) gene segments, and themouse exhibits a ratio of (a) B cells in the bone marrow that express animmunoglobulin having a λ, light chain, to (b) B cells in the bonemarrow that express an immunoglobulin having a κ light chain, of about 1to about 15. In some embodiments, the rearrangement includes a humanVκ1-39 gene segment. In some embodiments, the rearrangement includes ahuman Vκ3-20 gene segment. In some embodiments, the replacement of theendogenous immunoglobulin V_(H) gene segments is at an endogenousimmunoglobulin V_(H) locus. In some embodiments, the two human Vκ genesegments are at an endogenous immunoglobulin Vκ locus, and, in someembodiments, the two human Vκ gene segments replace all or substantiallyall mouse immunoglobulin Vκ gene segments. In some embodiments, the twohuman Vκ gene segments are at an endogenous immunoglobulin Vκ locus,and, in some embodiments, the two human Vκ gene segments replace all orsubstantially all mouse immunoglobulin Vκ and Jκ gene segments. Invarious embodiments, the two human Vκ gene segments are operably linkedto two or more (e.g., 2, 3, 4, 5) human Jκ gene segments.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and the ratio of immature B cells in the bonemarrow that express an immunoglobulin having a λ, light chain toimmature B cells that express an immunoglobulin having a κ light chainis about 1 to about 13.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and the ratio of mature B cells in the bonemarrow that express an immunoglobulin having a λ, light chain toimmature B cells that express an immunoglobulin having a κ light chainis about 1 to about 7.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a pro B cell population in the bonemarrow within in the range of about 2.5×10⁴ to about 1.5×10⁵ cells,inclusive, for example about 2.5×10⁴, 3.0×10⁴, 3.5×10⁴, 4.0×10⁴,4.5×10⁴, 5.0×10⁴, 5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴, 8.0×10⁴,8.5×10⁴, 9.0×10⁴, 9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 2.88×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 6.42×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 9.16×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 1.19×10⁵ cells. Exemplary pro Bcells in the bone marrow of genetically modified mice as describedherein are characterized by expression of CD19, CD43, c-kit and/or acombination thereof (e.g., CD19⁺, CD43⁺, c-kit⁺).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a pre B cell population in the bonemarrow within in the range of about 1×10⁶ to about 2×10⁶ cells,inclusive, for example, about 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶,1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or 2.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a pre Bcell population in the bone marrow of about 1.25×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 1.46×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 1.64×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 2.03×10⁶ cells. Exemplary pre Bcells in the bone marrow of genetically modified mice as describedherein are characterized by expression of CD19, CD43, c-kit and/or acombination thereof (e.g., CD19⁺, CD43⁻, c-kit⁻).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has an immature B cell population in thebone marrow within the range of about 5×10⁵ to about 7×10⁵ cells,inclusive, for example, about 5.0×10⁵, 5.1×10⁵, 5.2×10⁵, 5.3×10⁵,5.4×10⁵, 5.5×10⁵, 5.6×10⁵, 5.7×10⁵, 5.8×10⁵, 5.9×10⁵, 6.0×10⁵, 6.1×10⁵,6.2×10⁵, 6.3×10⁵, 6.4×10⁵, 6.5×10⁵, 6.6×10⁵, 6.7×10⁵, 6.8×10⁵, 6.9×10⁵,or 7.0×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.33×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.80×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.92×10⁵ cells; in some embodiments, the mouse comprises an immature Bcell population in the bone marrow of about 6.67×10⁵ cells. Exemplaryimmature B cells in the bone marrow of genetically modified mice asdescribed herein are characterized by expression of IgM, B220 and/or acombination thereof (e.g., IgM⁺, B220^(int)).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a mature B cell population in thebone marrow within the range of about 3×10⁴ to about 1.5×10⁵ cells,inclusive, for example about 3.0×10⁴, 3.5×10⁴, 4.0×10⁴, 4.5×10⁴,5.0×10⁴, 5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴, 8.0×10⁴, 8.5×10⁴,9.0×10⁴, 9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in some embodiments, amouse of the present invention comprises a mature B cell population inthe bone marrow of about 3.11×10⁴ cells; in some embodiments, a mouse ofthe present invention comprise a mature B cell population in the bonemarrow of about 1.09×10⁵ cells; in some embodiments, a mouse of thepresent invention comprises a mature B cell population in the bonemarrow of about 1.16×10⁵ cells; in some embodiments, a mouse of thepresent invention comprises a mature B cell population in the bonemarrow of about 1.44×10⁵ cells. Exemplary mature B cells in the bonemarrow of genetically modified mice as described herein arecharacterized by expression of IgM, B220 and/or a combination thereof(e.g., IgM⁺, B220^(hi)).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a total B cell population in the bonemarrow within the range of about 1×10⁶ to about 3×10⁶ cells, inclusive,for example about 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶,1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶,2.4×10⁶, 2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶ or 2.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a totalB cell population in the bone marrow of about 1.59×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 1.75×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 2.13×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 2.55×10⁶ cells. An exemplarytotal B cells in the bone marrow of genetically modified mice asdescribed herein are characterized by expression CD19, CD20 and/or acombination thereof (e.g., CD19⁺).

In some embodiments, a genetically modified mouse is provided thatexpresses an immunoglobulin light chain comprising a rearranged humanimmunoglobulin Vκ/Jκ sequence, wherein the mouse comprises a functionalimmunoglobulin λ, light chain locus, and wherein the mouse comprises asplenic B cell population that comprises a ratio of Igλ⁺ B cells to Igκ⁺B cells that is about 1 to about 8; in some embodiments, about 1 toabout 5. In some embodiments, the rearranged human immunoglobulin Vκ/Jκsequence is generated through a rearrangement of one of two humanimmunoglobulin Vκ gene segments and one of 1, 2, 3, 4, or 5 humanimmunoglobulin Jκ gene segments. In some embodiments, the rearrangedhuman immunoglobulin Vκ/Jκ sequence is generated through a rearrangementof a human immunoglobulin Vκ1-39 gene segment and a human immunoglobulinJκ gene segment selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combinationthereof. In some embodiments, the rearranged human immunoglobulin Vκ/Jκsequence is generated through a rearrangement of a human immunoglobulinVκ3-20 gene segment and a human immunoglobulin Jκ gene segment selectedfrom Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof.

In some embodiments, a mouse of the present invention comprises a CD19⁺splenic B cell population within the range of about 2×10⁶ to about 7×10⁶cells, inclusive, for example about 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a CD19⁺splenic B cell population of about 2.74×10⁶ cells; some embodiments, amouse of the present invention comprises a CD19⁺ splenic B cellpopulation of about 4.30×10⁶ cells; in some embodiments, a mouse of thepresent invention comprises a CD19⁺ splenic B cell population of about5.53×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺ splenic B cell population of about 6.18×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population within the range of about1×10⁶ to about 4×10⁶ cells, inclusive, for example about 1.0×10⁶,1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶ cells; in someembodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 1.30×10⁶; in someembodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 2.13×10⁶ cells; insome embodiments, a mouse of the present invention comprises CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 3.15×10⁶ cells; insome embodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 3.93×10⁶ cells.

In some embodiment, a mouse of the present invention comprises a CD19⁺,IgD^(lo), IgM^(hi) splenic B cell population within the range of about9×10⁵ to about 2×10⁶ cells, inclusive, for example about 9.0×10⁵,9.25×10⁵, 9.5×10⁵, 9.75×10⁵, 1.0×10⁶, 1.25×10⁶, 1.50×10⁶, 1.75×10⁶,2.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about9.52×10⁵; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.23×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.40×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.42×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and two or more(e.g., 2, 3, 4, or 5) unrearranged human Jκ gene segments, and whereinthe mouse comprises a peripheral splenic B cell population comprisingtransitional (e.g., T1, T2 and T3) B cell populations that are about thesame as a mouse that comprises a wild type complement of immunoglobulinκ light chain V and J gene segments. Exemplary transitional B cellpopulations (e.g., T1, T2 and T3) in the spleen of a geneticallymodified mouse as described herein are characterized by expression ofIgM, CD23, CD93, B220 and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises a T1 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(hi), CD23⁻)within the range of about 2×10⁶ to about 7×10⁶ cells, inclusive, forexample about 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶, 4.5×10⁶,5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T1 B cellpopulation in the spleen of about 2.16×10⁶ cells; in some embodiments, amouse of the present invention comprises a T1 B cell population in thespleen of about 3.63×10⁶ cells; in some embodiments, a mouse of thepresent invention comprises a T1 B cell population in the spleen ofabout 3.91×10⁶; in some embodiments, a mouse of the present inventioncomprises a T1 B cell population in the spleen of about 6.83×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a T2 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(hi), CD23⁺)within the range of about 1×10⁶ to about 7×10⁶ cells, inclusive, forexample about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells;in some embodiments, a mouse of the present invention mouse comprises aT2 B cell population in the spleen of about 1.30×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T2 B cellpopulation in the spleen of about 2.46×10⁶ cells; in some embodiments, amouse of the present invention comprises a T2 B cell population in thespleen of about 3.24×10⁶; in some embodiments, a mouse of the presentinvention comprises a T2 B cell population in the spleen of about6.52×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a T3 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(lo), CD23⁺)within the range of about 1×10⁶ to about 4×10⁶ cells, inclusive, forexample about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, or4.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a T3 B cell population in the spleen of about 1.08×10⁶ cells;in some embodiments, a mouse of the present invention comprises a T3 Bcell population in the spleen of about 1.35×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T3 B cellpopulation in the spleen of about 3.37×10⁶; in some embodiments, a mouseof the present invention comprises a T1 B cell population in the spleenof about 3.63×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and 1, 2, 3, 4,or 5 unrearranged human immunoglobulin Jκ gene segments, and wherein themouse comprises a peripheral splenic B cell population comprisingmarginal zone and marginal zone precursor B cell populations that areabout the same as a mouse that comprises a wild type complement ofimmunoglobulin Vκ and Jκ gene segments. Exemplary marginal zone B cellpopulations in the spleen of a genetically modified mouse as describedherein are characterized by expression of IgM, CD21/35, CD23, CD93, B220and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises marginalzone B cell population in the spleen (e.g., CD93⁻, B220⁺, IgM^(hi),CD21/35^(hi), CD23⁻) within the range of about 1×10⁶ to about 3×10⁶cells, inclusive, for example, about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶,or 3.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about1.47×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about1.49×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about2.26×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about2.33×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and 1, 2, 3, 4,or 5 unrearranged human immunoglobulin Jκ gene segments, and wherein themouse comprises a peripheral splenic B cell population comprisingfollicular (e.g., FO-I and FO-II) B cell population(s) that are aboutthe same as a mouse that comprises a wild type complement ofimmunoglobulin Vκ and Jκ gene segments. Exemplary follicular B cellpopulations (e.g., FO-I and FO-II) in the spleen of a geneticallymodified mouse as described herein are characterized by expression ofIgM, IgD, CD21/35, CD93, B220 and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises afollicular type 1 B cell population in the spleen (e.g., CD93⁻, B220⁺,CD21/35^(int), IgM^(lo), IgD^(hi)) within the range of about 3×10⁶ toabout 1.5×10⁷ cells, inclusive, for example about 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, 7.0×10⁶, 7.5×10⁶,8.0×10⁶, 8.5×10⁶, 9.0×10⁶, 9.5×10⁶, 1.0×10⁷, or 1.5×10⁷ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 3.57×10⁶ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 6.31×10⁶ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 9.42×10⁶ cells; in someembodiments, a mouse of the present invention comprise a follicular type1 B cell population in the spleen of about 1.14×10⁷ cells.

In some embodiments, a mouse of the present invention comprises afollicular type 2 B cell population in the spleen (e.g., CD93⁻, B220⁺,CD21/35^(int), IgM^(int), IgD^(hi)) within the range of about 1×10⁶ toabout 2×10⁶ cells, inclusive, for example, 1.0×10⁶, 1.25×10⁶, 1.5×10⁶,1.75×10⁶, or 2.0×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.14×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.45×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.80×10⁶; in some embodiments, a mouse of the present inventioncomprises a follicular type 2 B cell population in the spleen of about2.06×10⁶ cells.

In some other various aspects, also provided herein is a geneticallymodified non-human animal comprising in its germline an immunoglobulinlight chain locus comprising at least one human V_(L) gene segment andat least one human J_(L) gene segments operably linked to animmunoglobulin light chain constant region sequence, wherein each of theat least one human V_(L) gene segment comprises at least one histidinecodon that is not encoded by the corresponding human germline V_(L) genesegment. In one embodiment, the at least one human V_(L) gene segmentand the at least one human J_(L) gene segment are capable of rearrangingand encoding a human light chain variable domain of an antibody. In oneembodiment, the non-human animal does not comprise an endogenous V_(L)gene segment that is capable of rearranging to form an immunoglobulinlight chain. In one embodiment, the immunoglobulin light chain constantregion sequence is a non-human light chain constant region sequence,e.g., an endogenous immunoglobulin light chain constant region sequence,e.g., a rat or a mouse sequence. In one embodiment, the animal furthercomprises in its germline an immunoglobulin heavy chain locus thatcomprises an unrearranged heavy chain variable region sequencecomprising human V_(H), D_(H), and J_(H) gene segments operably linkedto an immunoglobulin heavy chain constant region sequence. In oneembodiment, the immunoglobulin heavy chain constant region sequence is anon-human immunoglobulin heavy chain constant region sequence, e.g., anendogenous non-human heavy chain constant region sequence, e.g., a rator a mouse sequence. In one embodiment, the at least one human V_(L)gene segment and the at least one human J_(L) gene segment are presentat the endogenous immunoglobulin light chain locus. In one embodiment,the immunoglobulin light chain constant region is a Cκ region. In oneembodiment, the at least one human V_(L) gene segments comprises asubstitution of at least one non-histidine codon encoded by acorresponding human germline V_(L) gene segment sequence with thehistidine codon. In one embodiment, the substitution is in the CDR3codon(s), e.g., three or four non-histidine codons. In one embodiment,the at least one human V_(L) gene segment is two human V_(L) genesegments, e.g., human Vκ1-39 and Vκ3-20 gene segments. In oneembodiment, the animal is a rodent, e.g., a rat or a mouse. In oneembodiment, the animal expresses an antibody comprising an amino acidsequence encoded by the at least one human V_(L) gene segments and theantibody retains at least one histidine residue at an amino acidposition encoded by the at least one histidine codon of the human V_(L)gene segment.

In one embodiment, also provided herein is a genetically modifiednon-human animal that comprises in its germline an immunoglobulin lightchain locus comprising a limited repertoire of human light chainvariable region gene segments, e.g., a limited repertoire of human V_(L)and J_(L) gene segments, wherein the limited repertoire of human lightchain variable gene segments comprises at least one histidine codon thatis not encoded by the corresponding human germline sequence. In oneembodiment, provided herein is a genetically modified non-human animalcomprising in its germline an immunoglobulin light chain locuscomprising no more than two human V_(L) gene segments and one or more,e.g., two or more, human J_(L) gene segments, wherein each of the nomore than two human V_(L) gene segments comprises at least one histidinecodon that is not encoded by the corresponding human germline V_(L) genesegment. In one embodiment, the no more than two human V_(L) genesegments and the one or more, e.g., the two or more, human J_(L) genesegments are operably linked to an immunoglobulin light chain constantregion sequence. In one embodiment, the no more than two human V_(L)gene segments and the one or more, e.g., the two or more, human J_(L)gene segments are Vκ and Jκ gene segments. In various embodiments, thehuman V_(L) gene segments and the human J_(L) gene segments are capableof rearranging and encoding a human light chain variable domain of anantibody. In one embodiment, the animal does not comprise an endogenousV_(L) gene segment that is capable of rearranging to form animmunoglobulin light chain. In one embodiment, the immunoglobulin lightchain constant region sequence is a non-human immunoglobulin constantregion sequence, e.g., a rodent sequence, e.g., a mouse or a ratsequence. In one embodiment, the non-human immunoglobulin light chainconstant region sequence is an endogenous sequence. In anotherembodiment, the immunoglobulin light chain constant region sequence is ahuman sequence. In one embodiment, the immunoglobulin light chainconstant region sequence is a Cκ sequence. In one embodiment, thenon-human animal further comprises in its germline an immunoglobulinheavy chain locus that comprises an unrearranged immunoglobulin heavychain variable region sequence that comprises human V_(H), D_(H), andJ_(H) gene segments operably linked to an immunoglobulin heavy chainconstant region sequence. In one embodiment, the immunoglobulin heavychain constant region sequence is a non-human immunoglobulin heavy chainconstant region sequence, e.g., a rodent sequence, e.g., a rat or amouse sequence. In one embodiment, the non-human immunoglobulin heavychain constant region sequence is an endogenous non-human immunoglobulinheavy chain constant region sequence. In one embodiment the heavy chainconstant region sequence is a human immunoglobulin heavy chain constantregion sequence. In one embodiment, the no more than two human V_(L)gene segments and the one or more, e.g., the two or more, human J_(L)gene segments are present at the endogenous non-human immunoglobulinlight chain locus.

In one embodiment, the non-human animal of the present inventioncomprises one or more, e.g., two or more human J_(L) gene segments, andthe one or more, e.g., two or more, human J_(L) gene segments are fivehuman Jκ segments, e.g., human Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 genesegments. In one embodiment, the no more than two human V_(L) genesegments are selected from human Vκ1-39 and Vκ3-20 gene segments, and acombination thereof. In one embodiment, the no more than two human V_(L)gene segments are human Vκ1-39 and Vκ3-20 gene segments. In oneembodiment, each of the no more than two V_(L) gene segments comprises asubstitution of at least one non-histidine codon encoded by thecorresponding human germline V_(L) segment sequence with a histidinecodon. In one embodiment, the substitution is of one, two, three, orfour codons (e.g., three or four codons). In one embodiment, thesubstitution is in the CDR3 codon(s). In the embodiment wherein the nomore than two human V_(L) gene segments are human Vκ1-39 and Vκ3-20 genesegments, each of the human Vκ1-39 and Vκ3-20 gene segments comprises asubstitution of at least one non-histidine codon encoded by acorresponding human germline V_(L) gene segment with the histidinecodon. In one embodiment, each of the human Vκ1-39 and Vκ3-20 genesegments comprises a substitution of three or four histidine codons. Inone embodiment, the three or four substitutions are in the CDR3 region.In one embodiment, wherein the substitution is of three non-histidinecodons of the human Vκ1-39 gene segment, the substitution is designed toexpress histidines at positions 106, 108, and 111. In anotherembodiment, wherein the substitution is of four non-histidine codons ofthe human Vκ1-39 gene segment, the substitution is designed to expresshistidines at positions 105, 106, 108, and 111. In another embodiment,wherein the substitution is of three non-histidine codons of the humanVκ3-20 gene segment, the substitution is designed to express histidinesat positions 105, 106, and 109. In yet another embodiment, wherein thesubstitution is of four non-histidine codons of the human Vκ3-20 genesegment, the substitution is designed to express histidines at positions105, 106, 107, and 109. In one embodiment, the non-human animal is arodent, e.g., a mouse or a rat. In one embodiment, the non-human animalis a mouse. In one embodiment, the animal expresses an antibodycomprising an amino acid sequence encoded by one of the no more than twohuman V_(L) gene segments and the antibody retains at least onehistidine residue at an amino acid position encoded by the at least onehistidine codon introduced into the human V_(L) gene segment. In oneembodiment, the animal expresses a population of antigen-specificantibodies in response to an antigen wherein all antibodies in thepopulation comprise (a) immunoglobulin light chain variable domainsderived from a rearrangement of the no more than two V_(L) gene segmentsand the one or more, e.g., the two or more, J_(L) gene segments whereineach of the no more than two human V_(L) gene segments comprises atleast one histidine codon that is not encoded by the corresponding humangermline V_(L) gene segment, and (b) immunoglobulin heavy chainscomprising human heavy chain variable domains derived from a repertoireof human heavy V, D, and J segments.

In one embodiment, the animal described herein comprises a population ofB cells in response to an antigen of interest that is enriched forantibodies that exhibit a decrease in dissociative half-life (t_(1/2))at an acidic pH as compared to neutral pH of at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 25-fold, or at least about 30-fold. In one embodiment, suchan enrichment in antibodies that exhibit a decrease in t_(1/2) is atleast about 2 fold.

Also provided herein is a method of generating an antibody that exhibitspH-dependent binding to an antigen of interest comprising generating thenon-human animal described herein (e.g., the non-human animal comprisingat least one human V_(L) gene segment and at least one human J_(L) genesegment; e.g., the non-human animal comprising a limited repertoire ofhuman light chain variable region gene segments; e.g., the non-humananimal comprising in its germline an immunoglobulin light chain locuscomprising no more than two human V_(L) gene segments and one or more,e.g., two or more, human J_(L) gene segments—wherein each of the humanV_(L) gene segments present in the germline of said animal comprises atleast one histidine codon that is not encoded by the corresponding humangermline V_(L) gene segment), immunizing said animal with an antigen ofinterest, and selecting an antibody that binds to the antigen ofinterest with a desired affinity at a neutral pH while displayingreduced binding to the antigen of interest at an acidic pH.

Also provided herein is a method of making a non-human animal thatcomprises a genetically modified immunoglobulin light chain locus in itsgermline, the method comprising (a) modifying a genome of the non-humananimal to delete or render non-functional endogenous immunoglobulinlight chain V_(L) and J_(L) gene segments in an immunoglobulin lightchain locus, and (b) placing in the genome of the non-human animal animmunoglobulin light chain variable region comprising at least one humanV_(L) gene segment and at least one human J_(L) gene segment, such thatthe immunoglobulin light chain variable region sequence is operablylinked to an immunoglobulin constant region sequence; wherein each ofthe at least one human V_(L) gene segments comprises at least onehistidine codon that is not encoded by the corresponding human germlineV_(L) gene segment. In one embodiment, the human V_(L) gene segment(s)and J_(L) gene segment(s) are capable of rearranging and encoding ahuman light chain variable domain of an antibody. In one embodiment, theimmunoglobulin light chain variable region is at the endogenousnon-human immunoglobulin light chain locus. In one embodiment, the atleast one human V_(L) gene segment is two human V_(L) gene segments, andwherein the two human V_(L) gene segments are human Vκ1-39 and Vκ3-20gene segments. In some embodiments, the non-human animal is a rodent,e.g., a mouse or a rat. In one embodiment, this method results in thenon-human animal that comprises a population of B cells enriched forantibodies exhibiting pH-dependent binding to an antigen of interest.

In some embodiments, also provided herein is a non-human immunoglobulinlight chain locus comprising at least one human V_(L) gene segment andat least one human J_(L) gene segment operably linked to animmunoglobulin constant region sequence, wherein each of the at leastone human V_(L) gene segments comprises at least one histidine codonthat is not encoded by the corresponding human germline V_(L) genesegment. In some embodiment, also provided is a non-human immunoglobulinlight chain locus comprising a limited repertoire of human variable genesegments, e.g., a non-human locus comprising no more than two humanV_(L) gene segments and one or more, e.g., two or more, human J_(L) genesegments operably linked to an immunoglobulin constant region sequence(e.g., a non-human immunoglobulin constant region sequence, e.g., a rator a mouse sequence), wherein each of the no more than two human V_(L)gene segments comprises at least one histidine codon that is not encodedby the corresponding human germline V_(L) gene segment. In oneembodiment, the locus comprises five human Jκ gene segments, e.g., Jκ1,Jκ2, Jκ3, Jκ4, and Jκ5 gene segments. In one embodiment, the no morethan two human V_(L) gene segments with histidine modifications areVκ1-39 and Vκ3-20. In various embodiments, the non-human loci describedherein may be generated using methods described throughout thisapplication for making genetically modified non-human animals. Thus, amethods of making genetically modified non-human animals comprising atleast one V_(L) gene segment and at least one J_(L) gene segment;comprising a limited repertoire of human variable gene segments; orcomprising no more than two human V_(L) gene segments and one or more,e.g., two or more, human J_(L) gene segments, operably linked to animmunoglobulin constant region sequence (e.g., a non-humanimmunoglobulin constant region sequence, e.g., a rat or a mousesequence), and wherein each human V_(L) gene segment comprises at leastone histidine codon that is not encoded by the corresponding humangermline V_(L) gene segment, is also provided.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an amino acid alignment of human Vκ1-39-derived lightchains from various antigen-specific antibodies (A-K antibodies,corresponding to SEQ ID NOs:136-146, respectively). Histidine (H)residues located within each light chain sequence are in bold. Variouslight chain regions (Framework and CDR) are indicated above thealignment.

FIG. 2 illustrates the combinations and locations of histidine residuesengineered in the CDR3 region of human Vκ1-39-derived light chains bymutagenesis. Corresponding nucleic acid sequences are included.Histidine residues introduced through mutagenesis and correspondingnucleic acid residues are shown in bold. Amino acid positions (105, 106,etc.) are based on a unique numbering described in Lefranc et al. (2003)Dev. Comp. Immunol. 27:55-77, and can also be viewed on the website ofthe International Immunogenetics Information System (IMGT).

FIG. 3 illustrates the level of antibody expression in ng/mL detected inthe supernatants of CHO cells transfected with nucleic acids encodingfive (1-5) different heavy chains and Vκ1-39-derived light chains havinghistidine residues engineered at indicated locations (see X axis) in theCDR3.

FIG. 4 is a western blot showing expression of selected antigen-specifichuman antibodies containing histidine engineered light chains in CHOcell supernatants.

FIGS. 5A-5E show the binding kinetics for selected heavy chains (1-5)from antigen-specific antibodies paired with various histidineengineered light chains at a neutral (7.4) and acidic (5.75) pH. Variouskinetic parameters including k_(a), k_(d), K_(D), and t_(1/2) are shown.NB=no binding.

FIG. 6 shows kinetic parameters (K_(D) and t_(1/2)) for antibodiescomprising parental universal light chain or histidine-modifieduniversal light chain paired with indicated heavy chains (2, 3, and 6).Histidine substitutions lead to strong pH dependence in severalantibodies. Histidine substitutions were made in CDR3 to convert thesequence ₁₀₅QQSYSTP₁₁₁ (SEQ ID NO:3) to histidine modified CDR3 sequencein the parentheses. Note that NB=no binding detected (KD >10micromolar).

FIG. 7 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ1-39/Jκ5 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 8A-8B show a general strategy for construction of targetingvectors for engineering of histidine residues into a rearranged humanlight chain variable region sequence derived from Vκ1-39/Jκ5 variableregion for making a genetically modified mouse that expresses antibodiescontaining the modified human light chain. FIGS. 8C-8D show introductionof the targeting vector for ULC-H105/106/108/111 substitutions into EScells and generation of heterozygous mice from the same; while FIGS.8E-8F show introduction of the targeting vector for ULC-H106/108/111substitutions into ES cells and generation of heterozygous mice from thesame. The diagrams are not presented to scale. Unless indicatedotherwise, filled shapes and solid lines represent mouse sequence, emptyshapes and double lines represent human sequence.

FIG. 9 shows antiserum titers against immunogen from mice heterozygousfor histidine universal light chain (HULC) (with 4 Hissubstitutions—HULC 1927 mice; with 3 His substitutions—HULC 1930 mice)and wild type animals in a second bleed.

FIG. 10 is a comparison of the number of total antigen positive clonesand the number of antigen positive clones displaying pH sensitiveantigen binding obtained from hybridoma fusions from heterozygous HULC(1927 vs 1930) and WT mice. Figure includes data for two mice for eachmouse type (“mouse 1” and “mouse 2”).

FIGS. 11A-11C show sensorgrams from surface plasmon resonance bindingexperiments in which monoclonal antibodies (AA, BB, CC, DD, HH, GG, NN,and OO) from either heterozygous HULC or WT mice were allowed toassociate with the immunogen at neutral pH (pH 7.4) followed by a shiftto a buffer with pH of either 7.4 or 6.0 for the dissociation phase. Theindividual lines in each graph represent the binding responses atdifferent concentrations of the respective antibodies. All experimentswere carried out at 25° C. Dissociative half-life values (t½) are notedabove the respective sensorgrams, and fold change in t½ is included tothe right of each sensorgram. Antibodies AA, BB, CC, DD, HH, and GG werefrom heterozygous HULC 1927 mice using His-substituted light chain, NNis from heterozygous HULC 1927 mouse using WT light chain, and OO isfrom a WT mouse (See Table 4 for clarification).

FIG. 12 shows positions of histidine residues engineered in the CDR3region of human Vκ3-20-derived light chains by mutagenesis. Histidineresidues introduced through mutagenesis and corresponding exemplarynucleic acid residues are shown in bold. Amino acid positions (105, 106,etc.) are based on a unique numbering described in Lefranc et al. (2003)Dev. Comp. Immunol. 27:55-77, and can also be viewed on the website ofthe International Immunogenetics Information System (IMGT).

FIG. 13 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ3-20/Jκ1 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 14A-14B show a general strategy for construction of targetingvectors for the engineering of histidine residues into a rearrangedhuman light chain variable region sequence derived from Vκ3-20/Jκ1 lightchain variable region for making a genetically modified mouse thatexpresses antibodies containing the modified human light chain. FIG. 14Cshows introduction of the targeting vector forULC-Q105H/Q106H/Y107H/S109H substitutions into ES cells and generationof heterozygous mice from the same; while FIG. 14D shows introduction ofthe targeting vector for ULC-Q105H/Q106H/S109H substitutions into EScells and generation of heterozygous mice from the same. The diagramsare not presented to scale. Unless indicated otherwise, filled shapesand solid lines represent mouse sequence, empty shapes and double linesrepresent human sequence.

FIG. 15 is a general illustration of recombination of a V and a J genesegment of an immunoglobulin κ light chain allele in a mouse and thestructure of the light chain locus before rearrangement (top) and afterrearrangement (bottom). Rearrangement depicted here is only one ofseveral possible rearrangement events. The diagrams are not presented toscale.

FIG. 16A is a schematic representation of two universal light chainloci, one comprising a rearranged human Vκ1-39/Jκ5 variable regionsequence (top), and one comprising a rearranged human Vκ3-20/Jκ1variable region sequence (bottom). The diagrams are not presented toscale. Unless indicated otherwise, filled shapes represent mousesequence, and empty shapes represent human sequence. FIG. 16B showsexamples of two genetically modified dual light chain (DLC) loci. Thelocus on the top (DLC-5J) contains an engineered human DNA fragmentcontaining two human Vκ gene segments and five human Jκ gene segments.The locus on the bottom (DLC-1J) contains an engineered human DNAfragment containing two human Vκ gene segments and one human Jκ genesegment. Each locus is capable of rearranging to form a human Vκ regionoperably linked to an endogenous light chain constant region (e.g., aCκ). Immunoglobulin promoters (P, open arrow above locus), leader exons(L, short open arrows), and the two human Vκ gene segments (long openarrows), all flanked upstream (5′) by a neomycin cassette containing Frtrecombination sites are shown. Recombination signal sequences engineeredwith each of the human gene segments (Vκ and Jκ) are indicated by openovals juxtaposed with each gene segment. DLC-5J locus contains an RSSjuxtaposed with each of the five Jκ gene segments. In most embodiments,unless indicated otherwise, filled shapes and solid lines representmouse sequences, and open shapes and double lines represent humansequences. The diagrams are not presented to scale.

FIGS. 17A-17C show a general strategy for construction of a targetingvector for the engineering of an immunoglobulin kappa locus comprisingtwo human Vκ segments (hVκ1-39 and hVκ3-20) and one human Jκ segment(Jκ5), as well as mouse enhancers and IgκC arm. FIG. 17D showsintroduction of this targeting vector into ES cells and generation ofheterozygous mice with the same; while FIG. 17E shows deletion of theselection cassette in ES cells using FLP enzyme. In most embodiments,unless indicated otherwise, filled shapes and solid lines representmouse sequences, and open shapes and double lines represent humansequences. The diagrams are not presented to scale.

FIGS. 18A-18D show the nucleotide sequence (SEQ ID NO:82) of theengineered portion of immunoglobulin κ locus comprising two human Vκsegments (hVκ1-39 and hVκ3-20) and one human Jκ segment; the nucleotidesequence spans the engineered human sequence and comprising about 100base pairs of endogenous mouse sequence at both the 5′ and the 3′ end.Bottom of FIG. 18D explains different fonts used to depict varioussequences.

FIGS. 19A-19B show a general strategy for construction of a targetingvector for the engineering of an immunoglobulin kappa locus comprisingtwo human Vκ segments (hVκ1-39 and hVκ3-20) and five human Jκ segments,as well as mouse enhancers and IgκC arm. FIG. 19C shows introduction ofthis targeting vector into ES cells and generation of heterozygous micewith the same; while FIG. 19D shows deletion of the selection cassettein ES cells using FLP enzyme. In most embodiments, unless indicatedotherwise, filled shapes and solid lines represent mouse sequences, andopen shapes and double lines represent human sequences. The diagrams arenot presented to scale.

FIGS. 20A-20D show the nucleotide sequence (SEQ ID NO:83) of theengineered immunoglobulin κ locus comprising two human Vκ segments(hVκ1-39 and hVκ3-20) and five human Jκ segments; the nucleotidesequence spans the engineered sequence and about 100 base pairs ofendogenous mouse sequence at both the 5′ and the 3′ end. Bottom of FIG.20D explains different fonts used to depict various sequences.

FIG. 21A, in the top panel, shows representative contour plots of bonemarrow stained for B and T cells (CD19⁺ and CD3⁺, respectively) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). The bottom panel shows representativecontour plots of bone marrow gated on CD19⁺ and stained for ckit⁺ andCD43⁺ from a wild type mouse (WT) and a mouse homozygous for two humanVκ and five human Jκ gene segments (DLC-5J). Pro and Pre B cells arenoted on the contour plots of the bottom panel.

FIG. 21B shows the number of Pro (CD19⁺CD43⁺ckit⁺) and Pre(CD19⁺CD43⁻ckit⁻) B cells in bone marrow harvested from the femurs ofwild type mice (WT) and mice homozygous for two human Vκ and five humanJκ gene segments (DLC-5J).

FIG. 22A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 22B shows the total number of B (CD19⁺), immature B(B220^(int)IgM⁺) and mature B (B220^(hi)IgM⁺) cells in bone marrowisolated from the femurs of wild type mice (WT) and mice homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 23A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 23B shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained forIgλ and Igκ expression isolated from the femurs of a wild type mouse(WT) and a mouse homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 24A, in the top panel, shows representative contour plots ofsplenocytes gated on singlets and stained for B and T cells (CD19⁺ andCD3⁺, respectively) from a wild type mouse (WT) and a mouse homozygousfor two human Vκ and five human Jκ gene segments (DLC-5J). The bottompanel shows representative contour plots of splenocytes gated on CD19+and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). Mature (54 for WT, 56.9 for DLC-5J) andtransitional (23.6 for WT, 25.6 for DLC-5J) B cells are noted on each ofthe contour plots.

FIG. 24B shows the total number of CD19⁺ B cells, transitional B cells)(CD19⁺IgM^(hi)IgD^(lo)) and mature B cells (CD19⁺IgM^(lo)IgD^(hi)) inharvested spleens from wild type mice (WT) and mice homozygous for twohuman Vκ and five human Jκ gene segments (DLC-5J).

FIG. 25A shows representative contour plots of Igλ⁺ and Igκ⁺ splenocytesgated on CD19⁺ from a wild type mouse (WT) and a mouse homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 25B shows the total number of B cells (CD19⁺), Igκ⁺ B cells(CD19⁺Igκ⁺) and Igλ⁺ B cells (CD19⁺Igλ⁺ in harvested spleens from wildtype (WT) and mice homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 26A shows the peripheral B cell development in mice homozygous fortwo human Vκ and five human Jκ gene segments. The first (far left)contour plot shows CD93⁺ and B220⁺ splenocytes gated on CD19⁺ indicatingimmature (39.6) and mature (57.8) B cells. The second (top middle)contour plot shows IgM⁺ and CD23⁺ expression in immature B cellsindicating T1 (33.7; IgD⁻IgM⁺CD21^(lo)CD23⁻), T2 (21.2;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) and T3 (29.1) B cell populations. Thethird (bottom middle) contour plot shows CD21⁺ (CD35⁺) and IgM⁺expression of mature B cells indicating a small population (14.8) whichgive rise to marginal zone B cells and a second population (70.5) whichgives rise to follicular (FO) B cells. The fourth (top right) contourplot shows B220⁺ and CD23⁺ expression in mature B cells indicatingmarginal zone (90.5; MZ) and marginal zone precursor (7.3;IgM^(hi)IgD^(hi)CD21^(hi)CD23⁺) B cell populations. The fifth (bottomright) contour plot shows IgD⁺ and IgM⁺ expression in mature B cellsindicating FO-I (79.0; IgD^(hi)IgM^(lo)CD21^(mid)CD23⁺) and FO-II (15.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 26B shows the peripheral B cell development in wild type mice. Thefirst (far left) contour plot shows CD93⁺ and B220⁺ splenocytes gated onCD19⁺ indicating immature (31.1) and mature (64.4) B cells. The second(top middle) contour plot shows IgM⁺ and CD23⁺ expression in immature Bcells indicating T1 (28.5; IgD⁻IgM⁺CD21^(lo)CD23⁻), T2 (28.7; IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) and T3 (30.7) B cell populations. The third(bottom middle) contour plot shows CD21⁺ (CD35⁺) and IgM⁺ expression ofmature B cells indicating a small population (7.69) which give rise tomarginal zone B cells and a second population (78.5) which gives rise tofollicular (FO) B cells. The fourth (top right) contour plot shows B220⁺and CD23⁺ expression in mature B cells indicating marginal zone (79.9;MZ) and marginal zone precursor (19.4; IgM^(hi)IgD^(hi)CD21^(hi)CD23⁺) Bcell populations. The fifth (bottom right) contour plot shows IgD⁺ andIgM⁺ expression in mature B cells indicating FO-I (83.6;IgD^(hi)IgM^(lo)CD21^(mid)CD23⁺) and FO-II (13.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 27 shows the total number of transitional, marginal zone andfollicular B cell populations in harvested spleens of wild-type (WT) andmice homozygous for two human Vκ and five human Jκ gene segments(DLC-5J).

FIG. 28 shows the relative mRNA expression in bone marrow (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (Hκ) (human light chain of aVELOCIMMUNE™ mouse), wild type mice (WT), mice homozygous for two humanVκ gene segments and five human Jκ gene segments (DLC-5J) and micehomozygous for two human Vκ gene segments and one human Jκ gene segment(DLC-1 J). Signals are normalized to expression of mouse Cκ. ND: notdetected.

FIG. 29 shows the relative mRNA expression in whole spleens (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (Hκ) (human light chain of aVELOCIMMUNE™ mouse), wild type mice (WT), mice homozygous for two humanVκ gene segments and five human Jκ gene segments (DLC-5J) and micehomozygous for two human Vκ gene segments and one human Jκ gene segment(DLC-1 J). Signals are normalized to expression of mouse Cκ. ND: notdetected.

FIG. 30 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer four histidineresidues into CDR3's of human Vκ1-39 and Vκ3-20 light chain sequence.SEQ ID NOs for these primers used in the Sequence Listing are includedin the Table below. F=forward primer, R=reverse primer.

FIG. 31A shows introduction of a targeting vector comprising two humanVκ light chain segments each substituted with four histidine residues(****) and five human Jκ into ES cells and generation of heterozygousmice with the same; while FIG. 31B shows deletion of the selectioncassette in ES cells using FLPo enzyme. In most embodiments, unlessindicated otherwise, filled shapes and solid lines represent mousesequences, and open shapes and double lines represent human sequences.The diagrams are not presented to scale.

FIG. 32 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer three histidineresidues into CDR3's of human Vκ1-39 and Vκ3-20 light chain sequence.SEQ ID NOs for these primers used in the Sequence Listing are includedin the Table below. F=forward primer, R=reverse primer.

FIG. 33A shows introduction of a targeting vector comprising two humanVκ light chain segments each substituted with three histidine residues(***) and five human Jκ into ES cells and generation of heterozygousmice with the same; while FIG. 33B shows deletion of the selectioncassette in ES cells using FLPo enzyme. In most embodiments, unlessindicated otherwise, filled shapes and solid lines represent mousesequences, and open shapes and double lines represent human sequences.The diagrams are not presented to scale.

FIG. 34A shows alignment of amino acid sequence encoded by humangermline Vκ3-20 sequence (bottom sequence) with amino acid translationof exemplary IgM light kappa chain variable sequence expressed in amouse comprising two V kappa segments (Vκ3-20 and Vκ1-39), eachsubstituted with 3 histidine residues in CDR3 sequence (top sequence);the alignment shows IgM kappa chain variable sequence expressed in amouse that retained all three histidine substitutions introduced intothe germline sequence. FIG. 34B shows alignment of amino acid sequenceencoded by human germline Vκ1-39 sequence (bottom sequence in eachalignment) with amino acid translation of exemplary IgM light kappachain variable sequence expressed in a mouse comprising two V kappasegments (Vκ3-20 and Vκ1-39), each substituted with 3 histidine residuesin CDR3 sequence (top sequence in each alignment); top alignment showsIgM kappa chain variable sequence expressed in a mouse that retained allthree histidine modifications introduced into the germline sequence,bottom alignment shows IgM kappa chain variable sequence expressed in amouse that retained two out of three histidine modifications introducedinto the germline sequence. In some embodiments, histidine introducedinto the last position of the Vκ may be lost during V-J rearrangement.

DETAILED DESCRIPTION OF INVENTION Definitions

The present invention provides genetically modified non-human animals(e.g., mice, rats, rabbits, hamsters, etc.) that comprise in theirgenome, e.g., in their germline, nucleotide sequence(s) encoding humanantibody molecules that exhibit pH-dependent antigen binding, e.g., anucleotide sequence of immunoglobulin light chain comprising rearrangedhuman immunoglobulin light chain variable region sequence encodingantibodies that exhibit pH-dependent antigen binding, e.g., a nucleotidesequence of immunoglobulin light chain comprising a limited repertoireof human V_(L) and J_(L) gene segments that rearrange and encodeantibodies that exhibit pH-dependent antigen binding; embryos, cells,and tissues comprising the same; methods of making the same; as well asmethods of using the same. Unless defined otherwise, all terms andphrases used herein include the meanings that the terms and phrases haveattained in the art, unless the contrary is clearly indicated or clearlyapparent from the context in which the term or phrase is used.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (C_(L)). The heavychain and light chain variable domains can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each heavy and light chain variable domaincomprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).The term “high affinity” antibody refers to an antibody that has a K_(D)with respect to its target epitope about of 10⁻⁹ M or lower (e.g., about1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment,K_(D) is measured by surface plasmon resonance, e.g., BIACORE™; inanother embodiment, K_(D) is measured by ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different immunogens) or onthe same molecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstheavy chain specific for a tumor antigen and a second heavy chainspecific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI,FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.).Further, the second heavy chain variable domain can be substituted witha heavy chain variable domain having a different desired specificity.For example, a bispecific antibody with a first heavy chain specific fora tumor antigen and a second heavy chain specific for a toxin can bepaired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) toa tumor cell. Other exemplary bispecific antibodies include those with afirst heavy chain specific for an activating receptor (e.g., B cellreceptor, FcγRI, FcγRIIA, FcγRIIIA, FcαRI, T cell receptor, etc.) and asecond heavy chain specific for an inhibitory receptor (e.g., FcγRIIB,CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can beconstructed for therapeutic conditions associated with cell activation(e.g. allergy and asthma). Bispecific antibodies can be made, forexample, by combining heavy chains that recognize different epitopes ofthe same immunogen. For example, nucleic acid sequences encoding heavychain variable sequences that recognize different epitopes of the sameimmunogen can be fused to nucleic acid sequences encoding the same ordifferent heavy chain constant regions, and such sequences can beexpressed in a cell that expresses an immunoglobulin light chain. Atypical bispecific antibody has two heavy chains each having three heavychain CDRs, followed by (N-terminal to C-terminal) a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin lightchain that either does not confer epitope-binding specificity but thatcan associate with each heavy chain, or that can associate with eachheavy chain and that can bind one or more of the epitopes bound by theheavy chain epitope-binding regions, or that can associate with eachheavy chain and enable binding of one or both of the heavy chains to oneor both epitopes. Similarly, the term “trispecific antibody” includes anantibody capable of selectively binding three or more epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. A CDR can be somatically mutated(e.g., vary from a sequence encoded in an animal's germline), humanized,and/or modified with amino acid substitutions, additions, or deletions.In some circumstances (e.g., for a CDR3), CDRs can be encoded by two ormore sequences (e.g., germline sequences) that are not contiguous (e.g.,in an unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. A heavy chain variable domain is encoded by a variable region genesequence, which generally comprises V_(H), D_(H), and J_(H) segmentsderived from a repertoire of V_(H), D_(H), and J_(H) segments present inthe germline. Sequences, locations and nomenclature for V, D, and Jheavy chain segments for various organisms can be found in IMGTdatabase, the website of the International Immunogenetics InformationSystem (IMGT).

The term “identity” when used in connection with sequence, includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappaand lambda light chains and a VpreB, as well as surrogate light chains.Light chain variable domains typically include three light chain CDRsand four framework (FR) regions, unless otherwise specified. Generally,a full-length light chain includes, from amino terminus to carboxylterminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. Alight chain variable domain is encoded by a light chain variable regiongene sequence, which generally comprises V_(L) and J_(L) segments,derived from a repertoire of V and J segments present in the germline.Sequences, locations and nomenclature for V and J light chain segmentsfor various organisms can be found in IMGT database, www.imgt.org. Lightchains include those, e.g., that do not selectively bind either a firstor a second epitope selectively bound by the epitope-binding protein inwhich they appear. Light chains also include those that bind andrecognize, or assist the heavy chain with binding and recognizing, oneor more epitopes selectively bound by the epitope-binding protein inwhich they appear. Common or universal light chains include thosederived from a human Vκ1-39Jκ5 gene or a human Vκ3-20Jκ1 gene, andinclude somatically mutated (e.g., affinity matured) versions of thesame. Dual light chains (DLC) include those derived from a light chainlocus comprising no more than two human Vκ segments, e.g., a humanVκ1-39 gene segment and a human Vκ3-20 gene segment, and includesomatically mutated (e.g., affinity matured) versions of the same.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

The phrase “somatically mutated” includes reference to a nucleic acidsequence from a B cell that has undergone class-switching, wherein thenucleic acid sequence of an immunoglobulin variable region (e.g.,nucleotide sequence encoding a heavy chain variable domain or includinga heavy chain CDR or FR sequence) in the class-switched B cell is notidentical to the nucleic acid sequence in the B cell prior toclass-switching, such as, for example, a difference in a CDR orframework nucleic acid sequence between a B cell that has not undergoneclass-switching and a B cell that has undergone class-switching.“Somatically mutated” includes reference to nucleic acid sequences fromaffinity-matured B cells that are not identical to correspondingimmunoglobulin variable region sequences in B cells that are notaffinity-matured (i.e., sequences in the genome of germline cells). Thephrase “somatically mutated” also includes reference to animmunoglobulin variable region nucleic acid sequence from a B cell afterexposure of the B cell to an epitope of interest, wherein the nucleicacid sequence differs from the corresponding nucleic acid sequence priorto exposure of the B cell to the epitope of interest. The phrase“somatically mutated” refers to sequences from antibodies that have beengenerated in an animal, e.g., a mouse having human immunoglobulinvariable region nucleic acid sequences, in response to an immunogenchallenge, and that result from the selection processes inherentlyoperative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “operably linked” refers to a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence.

“Functional” as used herein, e.g., in reference to a functionalpolypeptide, includes a polypeptide that retains at least one biologicalactivity normally associated with the native protein. In anotherinstance, a functional immunoglobulin gene segment may include avariable gene segment that is capable of productive rearrangement togenerate a rearranged immunoglobulin gene sequence.

“Neutral pH” includes pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., physiological pH. “Acidic pH” includes pH of 6.0 or lower, e.g.,pH between about 5.0 and about 6.0, pH between about 5.75 and about 6.0,e.g., pH of endosomal or lysosomal compartments.

Engineered Histidine Residues in Immunoglobulin Light Chain Genes

The inventors have discovered that non-human animals that expressantibodies that are capable of binding to an antigen in a pH dependentmanner can be made by making modifications of an immunoglobulin lightchain variable region at one or more positions along the sequence of thelight chain. Methods of making modifications in the germline of anon-human animal so that the animal would express histidines in CDRs ofantibodies are described. In particular, methods for makingmodifications in an immunoglobulin light chain variable sequence in thegermline of the mouse are described. Variable region sequence, e.g., oflight chains, typically show somatic hypermutation along the variableregion sequence, and, in some cases, such mutations can result in asubstitution of histidine residues (see, e.g., FIG. 1). Such mutationscan even occur in complementary determining regions (CDRs), which arethe regions of variable domains responsible for antigen binding. In somecases, such mutations can result in antibodies that display pH-dependentantigen binding, e.g., reduced antigen binding at an acidic pH ascompared to antigen binding at a neutral pH. Such pH-dependent antigenbinding is desired because it may enable the antibody to bind to theantigen outside the cell, and, when internalized into an endosome,release the antigen and recycle back to the surface to bind anotherantigen, avoiding target-mediated clearance. Approaches for introducinghistidine residues to achieve this effect by using a random his-scanningmutagenesis to engineer pH-dependent binding properties in anti-IL-6Rantibodies have been reported (US 2011/0111406 A1). However, randommutagenesis of antibody residues may result in decreased affinity ofantibody to the antigen. A non-human animal genetically modified toexpress a histidine substitution in antibody sequence enables generationof high-affinity antibodies in response to an antigen of interest that,due to histidine modification(s), would also display pH-dependentantigen binding.

Thus, in various embodiments, provided herein is a genetically modifiednon-human animal (e.g., rodent, e.g., a mouse or a rat) that comprisesin its genome, e.g., in its germline, a human immunoglobulin light chainvariable region sequence comprising modifications that result in theanimal expressing antibodies capable of binding to antigens in apH-dependent manner. In one embodiment, the non-human animal comprisesmodifications in the human immunoglobulin light chain variable regionsequence (e.g., V_(L) and/or J_(L) segment sequence) that comprisesubstitutions in at least one non-histidine codon (e.g., at least onenon-histidine codon encoded by the corresponding human germline V_(L)and/or J_(L) segment sequence) with a histidine codon (in some cases,also may be referred to as “histidine substitution,” “histidine codonsubstitution,” or the like). In one embodiment, the animal comprises agermline modification that allows histidine substitutions in CDR1, CDR2,CDR3, N terminal, loop 4 and even framework regions of the light chainin order to increase yield of pH dependent antibodies. In oneembodiment, the animal comprises at least one substitution of anon-histidine codon (e.g., at least one non-histidine codon encoded bythe corresponding human germline V_(L) and/or J_(L) segment sequence)with a histidine codon in a nucleotide sequence of a complementarydetermining region (CDR; e.g., CDR1, CDR2, and/or CDR3) of a humanimmunoglobulin light chain. In one embodiment, the substitution is in aCDR3 codon. In one embodiment, the light chain is a κ light chain. Inone embodiment, the animal expresses an immunoglobulin light chain,e.g., a light chain CDR, e.g., a light chain CDR3, comprising asubstitution of at least one amino acid with a histidine. In anotherembodiment, the light chain is a λ, light chain. In yet anotherembodiment, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon in both κ and λ, lightchains.

Histidine residue is encoded by two different codons, CAT and CAC(deoxyribonucleic acid residues). Thus, a non-histidine codon may besubstituted with a CAT or a CAC. The substitution is engineered in acodon that in its germline configuration (i.e., non-somatically mutatedstate) does not encode a histidine residue. Thus, the nucleotidesequence comprises at least one histidine codon that is not encoded by acorresponding human germline light chain variable gene segment (e.g.,corresponding human germline V_(L) and/or J_(L) gene segment).

In various embodiments, the histidine modifications in the light chainsequence of the genetically modified non-human animal can be designed invarious ways. In some embodiments, histidine substitutions may belimited to those positions requiring only a single nucleotide change. Insome embodiments histidine modifications may be made in artificialsequences (e.g., artificial D_(H) segments, N additions of identifiedantibodies, etc.) and these artificial sequences may be inserted intothe light chain sequence.

In one embodiment a light chain is a universal light chain (also termeda common light chain). As described in U.S. patent application Ser. Nos.13/022,759, 13/093,156, 13/412,936, 13/488,628, and 13/798,310 (U.S.Application Publication Nos. 2011/0195454, 2012/0021409, 2012/0192300,2013/0045492, and 2013/0185821, all incorporated herein by reference), anon-human animal (e.g., a mouse) that selects a common light chain for aplurality of heavy chains has a practical utility. In variousembodiments, antibodies expressed in a non-human animal comprising onlya common light chain will have heavy chains that can associate andexpress with an identical or substantially identical light chain. Thisis particularly useful in making bispecific antibodies. For example,such an animal can be immunized with a first immunogen to generate a Bcell that expresses an antibody that specifically binds a first epitope.The animal (or an animal genetically the same) can be immunized with asecond immunogen to generate a B cell that expresses an antibody thatspecifically binds the second epitope. Variable heavy chain regions canbe cloned from the B cells and expressed with the same heavy chainconstant region and the same light chain (e.g., a common light chain) ina cell to make a bispecific antibody, wherein the heavy chain componentof the bispecific antibody has been selected by an animal to associateand express with the same light chain component. In various embodimentsdescribed, the variable regions of the genetically engineered mice arehuman variable regions.

In another embodiment, a light chain is derived from a restricted(limited) light chain variable segment repertoire, e.g., light chainvariable segment repertoire comprising no more than two human V_(L) genesegments (e.g., a dual light chain or “DLC”). As described in moredetail in U.S. patent application Ser. No. 13/798,455, published as U.S.Patent Application Publication No. 2013/0198880, such limited lightchain variable segment repertoire results in generation of limited lightchain repertoire that also aids in generation of antibody componentsuseful for making bispecific or other multispecific antibodies.

In some embodiments, the dual light chain mouse may exhibit a morediverse light chain repertoire. In some embodiments, the dual lightchain mouse allows greater yield of binding antibodies, and limitingdiversity at the same time increases successful pairing with heavychains generated in a mouse comprising a single rearranged light chainvariable region, e.g., a universal light chain mouse. In someembodiments, the light chains may themselves exhibit antigen bindingproperties. In some embodiments, the mouse may be induced to produceantibodies exhibiting antigen specificity that resides in their lightchain (e.g., by limiting the mouse's immunoglobulin heavy chainrepertoire, e.g., by replacing the mouse heavy chain locus with a locuscomprising a single rearranged heavy chain variable region). In someembodiments, antibodies produced in such animals will be specific for aparticular first epitope (e.g., effector antigens, cytotoxic molecules,Fc receptors, toxins, activating or inhibitory receptors, T cellmarkers, immunoglobulin transporters, etc.) through their light chainbinding. Such epitope-specific human light chains derived from duallight chain mouse may be co-expressed with human heavy chains derivedfrom a mouse with a limited light chain repertoire, e.g., a ULC mouse,wherein the heavy chain is selected based on its ability to bind asecond epitope (e.g., a second epitope on a different antigen).

Thus, previously mice were engineered that are capable of generatingimmunoglobulin light chains that will suitably pair with a ratherdiverse family of heavy chains, including heavy chains whose humanvariable regions depart from germline sequences, e.g., affinity maturedor somatically mutated variable regions. In various embodiments, themice are devised to pair human light chain variable domains with humanheavy chain variable domains that comprise somatic mutations, thusenabling a route to high affinity binding proteins suitable for use ashuman therapeutics.

The genetically engineered mouse, through the long and complex processof antibody selection within an organism, makes biologically appropriatechoices in pairing a diverse collection of human heavy chain variabledomains with a limited number of human light chain options. In order toachieve this, the mouse is engineered to present a limited number ofhuman light chain variable domain options in conjunction with a widediversity of human heavy chain variable domain options. Upon challengewith an immunogen, the mouse maximizes the number of solutions in itsrepertoire to develop an antibody to the immunogen, limited largely orsolely by the number or light chain options in its repertoire. Invarious embodiments, this includes allowing the mouse to achievesuitable and compatible somatic mutations of the light chain variabledomain that will nonetheless be compatible with a relatively largevariety of human heavy chain variable domains, including in particularsomatically mutated human heavy chain variable domains.

The engineered common light chain or limited light chain repertoire micedescribed in U.S. Application Publication Nos. 2011/0195454,2012/0021409, 2012/0192300, 2013/0045492, 2013/0185821 and 2013/0198880comprised nucleic acid sequences encoding a limited repertoire of lightchain options, e.g., common or universal light chain “ULC” thatcomprised a single rearranged human immunoglobulin light chain variableregion sequence, e.g., a light chain that comprised no more than twohuman V_(L) segments, e.g., a dual light chain “DLC” that comprised twohuman V_(L) segments. To achieve such limited repertoire, mice wereengineered to render nonfunctional or substantially nonfunctional theirability to make, or rearrange, a native mouse light chain variabledomain. In one aspect, this was achieved, e.g., by deleting the mouse'slight chain variable region gene segments. As previously described, theendogenous mouse locus can then be modified by exogenous suitable humanlight chain variable region gene segments of choice, operably linked tothe endogenous mouse light chain constant domain, in a manner such thatthe exogenous human variable region gene segments can combine with theendogenous mouse light chain constant region gene and form a rearrangedreverse chimeric light chain gene (human variable, mouse constant). Invarious embodiments, the light chain variable region is capable of beingsomatically mutated. In various embodiments, to maximize ability of thelight chain variable region to acquire somatic mutations, theappropriate enhancer(s) is retained in the mouse. In one aspect, inmodifying a mouse κ light chain locus to replace endogenous mouse κlight chain gene segments with human κ light chain gene segments, themouse κ intronic enhancer and mouse κ 3′ enhancer are functionallymaintained, or undisrupted.

Thus, provided was a genetically engineered mouse that expresses alimited repertoire of reverse chimeric (human variable, mouse constant)light chains associated with a diversity of reverse chimeric (humanvariable, mouse constant) heavy chains. In various embodiments, theendogenous mouse κ light chain gene segments are deleted and replacedwith a single (or two) rearranged human light chain region, operablylinked to the endogenous mouse Cκ gene. In various embodiments, theendogenous mouse κ light chain gene segments are deleted and replacedwith a single human V_(L) segment that is capable of rearranging with ahuman light chain J segment (selected from one or plurality of J_(L)segments) and encoding a human variable domain of an immunoglobulinlight chain, wherein the single V_(L) and one or a plurality of J_(L)segments are operably linked to endogenous mouse Cκ gene. In othervarious embodiments, the endogenous mouse κ light chain gene segmentsare deleted and replaced with no more than two human V_(L) segments thatare capable or rearranging with a human light chain J segment (selectedfrom one or plurality of J_(L) segments, e.g., two or more J_(L)segments) and encoding a human variable domain of an immunoglobulinlight chain, wherein the no more than two V_(L) gene segments and one ora plurality of J_(L) segments are operably linked to endogenous mouse Cκgene. In other embodiments, the kappa light chain gene segments (e.g.,human V_(L) and J_(L) gene segments) are operably linked to human Cκgene. In embodiments for maximizing somatic hypermutation of therearranged human light chain region, the mouse κ intronic enhancer andthe mouse κ 3′ enhancer are maintained. In various embodiments, themouse also comprises a nonfunctional λ, light chain locus, or a deletionthereof or a deletion that renders the locus unable to make a λ, lightchain. In some specific embodiments, the locus of the geneticallyengineered mice with restricted light chain repertoires is substantiallyidentical to the loci depicted in FIGS. 16A and 16B.

In genetically engineered mice that comprise nucleic acid sequencesencoding a limited repertoire of light chain options, e.g., ULC micethat comprise a single rearranged human immunoglobulin light chainvariable region sequence, e.g., a light chain that comprise no more thantwo human V_(L) segments, e.g., DLC mice that comprise two human V_(L)segments, the immunoglobulin light chain locus differs from thewild-type immunoglobulin light chain locus.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that at least one,and in some embodiments all, mouse V_(L) gene segments are replaced byone human V_(L) gene segment or no more than two human V_(L) genesegments. In some embodiments, a single human V_(L) gene segment ispresent in the germline rearranged to a human J_(L) gene segment. Insome embodiments, human V_(L) gene segments of a mouse are capable ofrearranging to one of two or more human J_(L) gene segments to encode animmunoglobulin V_(L) domain of an antibody. In some embodiments, humanV_(L) gene segment(s) of a light chain locus of a mouse as describedherein is/are operably linked to two or more (e.g., two, three, four, orfive) human J_(L) gene segments.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that it does notcontain a nucleotide sequence before rearrangement that encodes anendogenous V_(L) gene segment. In some embodiments, the structure of thelight chain locus of such mouse is different from that of the referencestructure of FIG. 15 in that it does not contain a nucleotide sequencebefore rearrangement that encodes an endogenous J_(L) gene segment. Insome embodiments, the structure of the light chain locus of such mouseis different from that of the reference structure of FIG. 15 in that itdoes not contain a nucleotide sequence before rearrangement that encodesendogenous V_(L) and J_(L) gene segments.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that it does notcontain a nucleotide sequence after rearrangement that encodes anendogenous V_(L) gene segment. In some embodiments, the structure of thelight chain locus of such a mouse is different from that of thereference structure of FIG. 15 in that it does not contain a nucleotidesequence after rearrangement that encodes an endogenous J_(L) genesegment. In some embodiments, the structure of the light chain locus ofsuch a mouse is different from that of the reference structure of FIG.15 in that it does not contain a nucleotide sequence after rearrangementthat encodes endogenous V_(L) and J_(L) gene segments.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that it contains nomore than two human V_(L) gene segments and one or more, e.g., two ormore (e.g., two, three, four, or five) human J_(L) gene segments beforerearrangement. In some embodiments, the light chain locus of such amouse is different from that of the reference structure of FIG. 15 inthat it contains no more than two human V_(L) gene segments and fivehuman J_(L) gene segments before rearrangement.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that it contains nomore than two human V_(L) gene segments and five or less (e.g., 5, 4, 3,2, or 1) human J_(L) gene segments after rearrangement. In someembodiments, the light chain locus of such a mouse is different fromthat of the reference structure of FIG. 15 in that contains no more thantwo human V_(L) gene segments and one, two, three, four, or five humanJ_(L) gene segments after rearrangement. In one embodiment, thestructure of the light chain locus of the mouse that comprises a nucleicacid sequences encoding a limited repertoire of light chain options(e.g., a ULC mouse, e.g., a DLC mouse) is different from that of thereference structure of FIG. 15 in that it contains one human V_(L) andfive or less (e.g., 5, 4, 3, 2, or 1) human J_(L) gene segments afterrearrangement.

In various embodiments, human V_(L) and J_(L) gene segments are human Vκand Jκ gene segments. In various embodiments, human Vκ segments areselected from a human Vκ1-39 gene segment and a human Vκ3-20 genesegment. In some embodiments, human Vκ segments are human Vκ1-39 andhuman Vκ3-20. In some embodiments, human Jκ segments are selected from aJκ1, Jκ2, Jκ3, Jκ4, Jκ5 gene segment, and a combination thereof. In someembodiments, human Jκ gene segments are human Jκ1, Jκ2, Jκ3, Jκ4, andJκ5.

In some embodiments, the structure of the light chain locus of the mousethat comprises a nucleic acid sequences encoding a limited repertoire oflight chain options (e.g., a ULC mouse, e.g., a DLC mouse) is differentfrom that of the reference structure of FIG. 15 in that it contains astructure that is substantially the same as that of the structure ofFIGS. 16A and 16B before rearrangement (e.g., structures in FIGS. 8C,8E, 14C, 14D, 17E, 19D, 31, and 33). In some embodiments, a mouse isprovided, comprising a light chain locus whose structure is identical tothe structure of FIGS. 16A and 16B before rearrangement.

Mice containing human immunoglobulin loci, variable and constant regionsrandomly inserted into the mouse genome, are known in the art. Initialstrains of such mice contained a limited number of human immunoglobulingene segments. Specifically, a handful of strains containing humanimmunoglobulin light chain gene segments contained either one, three orfour human immunoglobulin V_(L) gene segments and five humanimmunoglobulin J_(L) gene segments (Taylor et al. 1992, Nucleic AcidsResearch 20(23): 6287-6295; Fishwild et al. 1996, Nature Biotechnology14: 845-851; Lonberg et al. 1994, Nature 368: 856-859; Green et al.1994, Nature Genetics 7:13-21; Green and Jakobovits 1998, J. Exp. Med.188(3): 483-495; Green 1999, J. Immunol. Methods 231: 11-23). These micethat contained only a few human immunoglobulin V_(L) gene segments aspart of fully human transgenes randomly inserted into the mouse genomedemonstrated compromised B cell numbers, impaired B cell development andother immune deficiencies. Expression of the human immunoglobulinvariable region genes, as detected by surface expression of human Cκ onB cells, was lower than the endogenous κ light chain as compared to wildtype. Surprisingly, mice with limited repertoire of light chain optionssuch as mice engineered to contain at the endogenous immunoglobulin κlight chain loci either one or two human immunoglobulin Vκ genesegments, display B cell numbers and development that was nearlywild-type (see, e.g., U.S. Patent Application Publication No.2013/0198880 and present examples). Further, in some embodiments, thesemice are able to generate several high-affinity reverse chimericantibodies containing human variable light and heavy chain domains inresponse to antigen, wherein the variable light chain domains eachcontain one of two possible human V_(L) gene segments and one of fivepossible human J_(L) gene segments (see, e.g., U.S. Patent ApplicationPublication No. 2013/0198880, and present examples). Thus, in contrastto preliminary strains of mice engineered with human immunoglobulinlight chain miniloci (i.e., a limited number of human immunoglobulingene segments), mice that contain a limited number of humanimmunoglobulin V_(L) gene segments (either one or two) and, in someembodiments, two or more (e.g., 2, 3, 4, or 5) human immunoglobulinJ_(L) gene segments, surprisingly exhibit normal B cell numbers, normalimmunoglobulin light chain expression, and normal B cell development.Further, such mice also show no reduced or impaired ability to mountrobust immune responses to multiple antigens as a result of a limitedimmunoglobulin light chain repertoire. Accordingly, in some embodiments,mice that comprise a humanized variable light chain locus comprising nomore than two unrearranged human immunoglobulin V_(L) gene segments andtwo or more (e.g., 2, 3, 4, or 5) human immunoglobulin J_(L) genesegments—or no more than two rearranged human V_(L)J_(L)segments—exhibit wild-type B cell populations in number, and exhibitedwild-type B cell development.

The antibodies generated in the universal light chain mice or mice withlimited light chain repertoire (e.g., dual light chain mice) in responseto various antigens are capable of utilizing a diverse repertoire ofheavy chain variable region sequences, comprising a diverse repertoireof V_(H), D_(H), and J_(H) segments. Antibodies generated in suchgenetically engineered mice are useful for designing bispecifictherapeutic antibodies; however, as with any other antibody, eachbispecific antibody may only bind to one target during its lifetime inthe plasma; the antibody is internalized into an endosome and targetedfor lysosomal degradation. Studies have shown that MHC-class-I-like Fcγreceptor FcRn is capable of rescuing immunoglobulins from lysosomaldegradation by recycling it back to the cell surface from the sortingendosome. Simister and Mostov (1989) An Fc receptor structurally relatedto MHC class I antigens. Nature 337: 184-87. As explained above, toimprove efficiency of antibody recycling, further modifications toantibody sequences, e.g., modifications that result in decreased antigenbinding at acidic pH (e.g., pH of the endosome), while retainingantibody-antigen affinity and specificity at neutral pH (e.g.,physiological pH) are beneficial. The non-human animals describedherein, wherein histidine residues are substituted for non-histidineresidues in the light chain sequence are beneficial because they arecapable of producing high-affinity antibodies based on universal lightchain or restricted light chain repertoire (e.g., DLC) format that alsodisplay pH-dependent binding, e.g., display reduced binding to theantigen at acidic versus neutral pH.

Thus, in one embodiment, provided herein is a non-human animal (e.g., arodent, e.g., a mouse or a rat) that comprises in its genome, e.g., inits germline, a limited repertoire of human light chain variableregions, or a single human light chain variable region, from a limitedrepertoire of human light chain variable gene segments, wherein thehuman light chain variable region(s) comprise at least one substitutionof a non-histidine codon for a histidine codon. In some embodiments,provided non-human animals are genetically engineered to include asingle unrearranged human light chain variable region gene segment (ortwo human light chain variable region gene segments) that rearranges toform a rearranged human light chain variable region gene (or tworearranged light chain variable region genes) that expresses a singlelight chain (or that express either or both of two light chains),wherein the light chain variable region gene(s) comprise a substitutionof at least one non-histidine codon with a histidine codon. Therearranged human light chain variable domains encoded by thesehistidine-substituted light chain variable region gene(s) are capable ofpairing with a plurality of affinity-matured human heavy chains selectedby the animals, wherein the heavy chain variable regions specificallybind different epitopes. In various embodiments, the at least onesubstitution of a non-histidine residue with a histidine residue resultsin a rearranged human light chain that, when expressed with a cognateheavy chain, binds to its antigen in a pH-dependent manner.

Genetically engineered animals are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region gene sequences, wherein the variable region genesequences comprise at least one substitution of a non-histidine codonwith a histidine codon. In some embodiments, provided animals aregenetically engineered to include a single V/J human light chainsequence (or two V/J sequences) that comprises a substitution of atleast one non-histidine codon with a histidine codon and expresses avariable region of a single light chain (or that express either or bothof two variable regions). In one aspect, a light chain comprising thevariable sequence is capable of pairing with a plurality ofaffinity-matured human heavy chains clonally selected by the animal,wherein the heavy chain variable regions specifically bind differentepitopes. In one embodiment, the antibody binds to its antigen(s) in apH-dependent manner. In one embodiment, the single V/J human light chainsequence is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the two V/J sequences are Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the Vκ1-39Jκ5 and Vκ3-20Jκ1 sequences are rearranged V/J sequences.

In one aspect, a genetically modified non-human animal is provided thatcomprises a single human immunoglobulin light chain V_(L) gene segmentthat is capable of rearranging with a human J_(L) gene segment (selectedfrom one or a plurality of J_(L) segments) and encoding a human variabledomain of an immunoglobulin light chain, wherein the single humanimmunoglobulin light chain V_(L) gene segment and/or human J_(L) genesegment comprise a substitution of at least one non-histidine codon witha histidine codon (e.g., substitution of at least one non-histidinecodon encoded by the corresponding human germline V_(L) and/or J_(L)gene segment with a histidine). In another aspect, a geneticallymodified mouse is provided that comprises no more than two human V_(L)gene segments, each of which is capable of rearranging with a humanJ_(L) gene segment (selected from one or a plurality of J_(L) segments)and encoding a human variable domain of an immunoglobulin light chain,wherein each of the no more than two V_(L) gene segments and/or theJ_(L) gene segment comprise a substitution of at least one non-histidinecodon with a histidine codon (e.g., substitution of at least onenon-histidine codon encoded by the corresponding human germline V_(L)and/or J_(L) gene segment with a histidine). In some certainembodiments, the no more than two human V_(L) gene segments are selectedfrom the group consisting of a human Vκ1-39 gene segment, a human Vκ3-20gene segment, and a combination thereof. In some certain embodiments,the no more than two human V_(L) gene segments are a human Vκ1-39 genesegment and a human Vκ3-20 gene segment.

In one aspect, a genetically modified mouse is provided that comprises asingle rearranged (V/J) human immunoglobulin light chain variable region(i.e., a V_(L)/J_(L) region) that encodes a human variable domain of animmunoglobulin light chain, wherein the single rearranged variableregion comprises a substitution of at least one non-histidine codon witha histidine codon. In another aspect, the mouse comprises no more thantwo rearranged human variable regions that are capable of encoding ahuman variable domain of an immunoglobulin light chain, wherein each ofthe no more than two rearranged variable regions comprise a substitutionof at least one histidine codon.

Thus, provided herein is a genetically modified non-human animal thatcomprises in its genome, e.g., in its germline, a single rearrangedhuman immunoglobulin light chain variable region sequence comprisinghuman V_(L) and J_(L) sequences wherein the single rearranged humanimmunoglobulin light chain variable region comprises a substitution ofat least one non-histidine codon with a histidine codon (e.g.,substitution of at least one non-histidine codon encoded by thecorresponding human germline V_(L) and/or J_(L) gene segment with ahistidine). In one aspect, the single rearranged human immunoglobulinlight chain variable region sequence is derived from human germlineV_(L) and J_(L) gene sequences, but for the histidine substitution(s).In one embodiment, the human immunoglobulin light chain is a humanimmunoglobulin κ chain. Thus, in one embodiment, the human V_(L) genesequence is selected from Vκ1-39 and Vκ3-20. In one embodiment, thesingle rearranged human immunoglobulin light chain variable regionsequence comprises rearranged Vκ1-39/J or Vκ3-20/J sequence. In oneembodiment, the human J_(L) gene sequence is selected from Jκ1, Jκ2,Jκ3, Jκ4, and Jκ5. In one embodiment the human J_(L) sequence isselected from Jκ1 and Jκ5. In one embodiment, the single rearrangedhuman immunoglobulin light chain variable region sequence is selectedfrom Vκ1-39Jκ5 and Vκ3-20Jκ1 (e.g., but for the histidinesubstitution(s)). In an alternative embodiment, the human immunoglobulinlight chain is a human λ, chain.

Also, in one embodiment, provided herein is a genetically modifiednon-human animal that comprises in its genome, e.g., in its germline, alimited repertoire, e.g., no more than two, unrearranged human V_(L)gene segments and one or more, e.g., two or more (e.g., 2, 3, 4, or 5),unrearranged human J_(L) gene segments wherein each unrearranged humanV_(L) and/or human J_(L) gene segment comprises substitution of at leastone non-histidine codon for a histidine codon, e.g., at least onenon-histidine codon present in the germline sequence for a histidinecodon. In another embodiment, provided herein is a genetically modifiednon-human animal that comprises in its genome, e.g., in its germline, alimited repertoire, e.g., no more than two, unrearranged human V_(L)gene segments and one or more, e.g., two or more (e.g., 2, 3, 4, or 5),unrearranged human J_(L) gene segments wherein each unrearranged humanV_(L) and, optionally, human J_(L) gene sequence(s), comprisessubstitution of at least one non-histidine codon for a histidine codon,e.g., at least one non-histidine codon present in the correspondinghuman germline sequence for a histidine codon. Thus, in one aspect, thevariable gene segment sequence in the germline of an animal is a humangermline V_(L) and/or J_(L) gene sequences, but for the histidinesubstitution(s). Histidine substitutions are positioned such that, uponrearrangement, the rearranged light chain sequence is designed tocontain a substitution of at least one non-histidine codon with ahistidine codon. In one embodiment, the human immunoglobulin light chainis a human immunoglobulin κ chain. Thus, in one embodiment, the humanV_(L) gene sequence is selected from Vκ1-39 and Vκ3-20. Thus, in oneembodiment, the genetically modified non-human animal comprises in itsgenome, e.g., in its germline, an unrearranged human Vκ1-39 andunrearranged human Vκ3-20 gene segments and one or more, e.g., two ormore, unrearranged human J_(L) segments (e.g., Jκ1, Jκ2, Jκ3, Jκ4,and/or Jκ5 gene segments), wherein the Vκ1-39 and Vκ3-20 gene segmentsare capable of rearranging with said human J_(L) segments, and whereineach of the variable region gene segment sequences present in thegermline comprise a substitution of at least one non-histidine codonwith a histidine codon, e.g., at least one non-histidine codon presentin the germline sequence with a histidine codon. In another embodiment,the genetically modified non-human animal comprises in its genome, e.g.,in its germline, an unrearranged human Vκ1-39 and unrearranged humanVκ3-20 gene segments and one or more, e.g., two or more, unrearrangedhuman J_(L) segments (e.g., Jκ1, Jκ2, Jκ3, Jκ4, and/or Jκ5 genesegments), wherein the Vκ1-39 and Vκ3-20 gene segments are capable ofrearranging with said human J_(L) segments, and wherein each of theVκ1-39 and Vκ3-20 gene segments comprise a substitution of at least onenon-histidine codon with a histidine codon, and, optionally, the J_(L)segments may also comprise histidine substitution(s). In one embodiment,the genetically modified non-human animal comprises Jκ1, Jκ2, Jκ3, Jκ4,and Jκ5 gene segments. Thus, in one embodiment, the V_(L) and,optionally, J_(L) sequences at the κ light chain locus are essentiallygermline sequences but for the histidine substitution(s).

In one embodiment, the substitution of at least one non-histidine codonfor a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR) of the light chain variabledomain. In one embodiment, the substitution of at least onenon-histidine codon for a histidine codon is in the nucleotide sequenceencoding CDR1, CDR2 or CDR3 of the light chain variable domain. In onespecific embodiment, the substitution is in the nucleotide sequenceencoding CDR3.

In one aspect, the substitution is of at least one non-histidine codonfor a histidine codon in the CDR3 codon of the human light chainvariable region gene sequence. In one embodiment, the substitution is ofone, two, three, four, or more CDR3 codons. In the embodiment whereinthe non-human animal comprises a single rearranged human immunoglobulinlight chain variable region that is a Vκ1-39Jκ5 variable region or thenon-human animal comprises no more than two unrearranged human V_(L)gene segments one of which is a Vκ1-39 gene segment, the replacement inthe Vκ1-39 sequence of at least one non-histidine codon with a histidinecodon comprises a replacement at a position in the immunoglobulin lightchain gene sequence encoding CDR3 designed to express a histidine atposition selected from 105, 106, 108, 111, and a combination thereof. Inone embodiment, the replacement is designed to express histidines atpositions 105 and 106. In one embodiment, the replacement is designed toexpress histidines at positions 105 and 111. In one embodiment, thereplacement is designed to express histidines at positions 105 and 108.In one embodiment, the replacement is designed to express histidines atpositions 105, 108 and 111. In one embodiment, the replacement isdesigned to express histidines at positions 105, 106, and 108. In oneembodiment, the replacement is designed to express histidines atpositions 106 and 108. In one embodiment, the replacement is designed toexpress histidines at positions 106 and 111. In one embodiment, thereplacement is designed to express histidines at positions 108 and 111.In one embodiment, the replacement is designed to express histidines atpositions 106, 108, and 111. In yet another embodiment, the replacementis designed to express histidines at positions 106, 108 and 111. In oneembodiment, the replacement is designed to express histidines atpositions 105, 106, and 111. In one embodiment, the replacement isdesigned to express histidines at positions 105, 106, 108, and 111. Inone embodiment, the nucleic acid and amino acid sequences of thehistidine-substituted CDR3 regions are depicted in sequence alignment ofFIG. 2 and set forth in SEQ ID NOs: 4-33. In one embodiment, wild typeCDR3 nucleic acid and amino acid sequences (depicted in FIG. 2) are setforth in SEQ ID NOs:2 and 3, respectively. Other embodiments of nucleicacid and amino acid sequences of histidine-substituted CDR3 sequencesappear throughout the specification and the Sequence Listing, and shouldbe clear to those skilled in the art.

In the embodiment wherein the non-human animal comprises a singlerearranged human immunoglobulin light chain variable region that is aVκ3-20Jκ1 variable region or the non-human animal comprises no more thantwo unrearranged human V_(L) gene segments one of which is a Vκ3-20 genesegment, the replacement in the Vκ3-20 sequence of at least onenon-histidine codon with a histidine codon comprises a replacement at aposition in the immunoglobulin light chain gene sequence encoding CDR3region that is designed to express a histidine at position selected from105, 106, 107, 109, and a combination thereof. In one embodiment, thereplacement is designed to express histidines at positions 105 and 106.In one embodiment, the replacement is designed to express histidines atpositions 105 and 107. In one embodiment, the replacement is designed toexpress histidines at positions 105 and 109. In one embodiment, thereplacement is designed to express histidines at positions 106 and 107.In one embodiment, the replacement is designed to express histidines atpositions 106 and 109. In one embodiment, the replacement is designed toexpress histidines at positions 107 and 109. In one embodiment, thereplacement is designed to express histidines at positions 105, 106, and107. In one embodiment, the replacement is designed to expresshistidines at positions 105, 107, and 109. In one embodiment, thereplacement is designed to express histidines at positions 106, 108, and111. In one embodiment, the replacement is designed to expresshistidines at positions 105, 106 and 109. In another embodiment, thereplacement is designed to express histidines at positions 105, 106,107, and 109. The nucleic acid and amino acid sequences of exemplaryhistidine-substituted CDR3 regions are depicted in sequence alignment ofFIG. 12 and set forth in SEQ ID NOs: 76-79. Wild type CDR3 nucleic acidand amino acid sequences (depicted in FIG. 12) are set forth in SEQ IDNOs:74 and 75, respectively. Other embodiments of nucleic acid and aminoacid sequences of histidine-substituted CDR3 sequences appear throughoutthe specification and the Sequence Listing, and should be clear to thoseskilled in the art.

Amino acid positions (105, 106, etc.) are based on a unique numberingdescribed in Lefranc et al. (2003) Dev. Comp. Immunol. 27:55-77, and canalso be viewed on www.imgt.org.

In one embodiment, the human V_(L) gene segment is operably linked to ahuman or non-human leader sequence. In one embodiment, the leadersequence is a non-human leader sequence. In a specific embodiment, thenon-human leader sequence is a mouse Vκ3-7 leader sequence. In aspecific embodiment, the leader sequence is operably linked to anunrearranged human V_(L) gene segment. In a specific embodiment, theleader sequence is operably linked to a rearranged human V_(L)/J_(L)sequence. Thus, in one specific embodiment, the single rearrangedVκ1-39/Jκ5 or Vκ3-20/Jκ1 variable region gene sequence comprising atleast one histidine substitution is operably linked to a mouse Vκ3-7leader sequence. In another specific embodiment, the unrearranged humanVκ1-39 and/or Vκ3-20 gene segments comprising at least one histidinesubstitution are operably linked to a mouse Vκ3-7 leader sequence. Inyet another embodiment, the unrearranged human Vκ1-39 and/or Vκ3-20 genesegments are operably lined to a human Vκ leader sequences. In oneembodiment, the unrearranged human Vκ1-39 gene segment comprising atleast one histidine substitution is linked to a human Vκ1-39 leadersequence, and the unrearranged human Vκ3-20 gene segment comprising atleast one histidine substitution is linked to a human Vκ3-20 leadersequence.

In one embodiment, the V_(L) gene segment is operably linked to animmunoglobulin promoter sequence. In one embodiment, the promotersequence is a human promoter sequence. In a specific embodiment, thehuman immunoglobulin promoter is a human Vκ3-15 promoter. In anotherspecific embodiment, the human immunoglobulin promoter is a human Vκ1-39or Vκ3-20 promoter. In a specific embodiment, the promoter is operablylinked to an unrearranged human V_(L) gene segment. In a specificembodiment, the promoter is operably linked to a rearranged humanV_(L)/J_(L) sequence. Thus, in one specific embodiment, the singlerearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 variable region gene sequencecomprising at least one histidine substitution is operably linked to thehuman Vκ3-15 promoter. In another specific embodiment, the unrearrangedhuman Vκ1-39 and/or Vκ3-20 gene segments comprising at least onehistidine substitution are each operably linked to the human Vκ3-15promoter. In another specific embodiment, the unrearranged human Vκ1-39and Vκ3-20 gene segments comprising at least one histidine substitutionare linked to human Vκ1-39 and Vκ3-20 promoter, respectively.

In one embodiment, the light chain locus comprises a leader sequence (a)flanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and (b) flanked 3′ with ahuman V_(L) gene segment that rearranges with a human J_(L) segment andcomprises substitution of at least one non-histidine codon with ahistidine codon; and the light chain locus encodes an immunoglobulinlight chain comprising a variable domain of a reverse chimeric lightchain and an endogenous non-human light chain constant region (C_(L)).In a specific embodiment, the V_(L) and J_(L) gene segments are at thenon-human Vκ locus, and the non-human C_(L) is a non-human Cκ (e.g.,mouse Cκ). In one specific embodiment, the variable region sequence isoperably linked to the non-human constant region sequence, e.g., thenon-human Cκ gene sequence. In one embodiment, the non-humanimmunoglobulin light chain constant region sequence is an endogenousnon-human sequence. In another specific embodiment, the C_(L) is a humanCκ. In one embodiment, the non-human animal is a mouse and the Cκ genesequence is a mouse Cκ gene sequence. In one embodiment, the human V_(L)gene segment that rearranges with a human J_(L) segment and comprisessubstitution of at least one non-histidine codon with a histidine codonis at the endogenous non-human (e.g., mouse) immunoglobulin light chainlocus (κ locus). Exemplary embodiments of the locus are presented inFIGS. 31A and 33A.

In one embodiment, the light chain locus comprises a leader sequence (a)flanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and (b) flanked 3′ with arearranged human variable region sequence (V_(L)/J_(L) sequence)comprising a substitution of at least one non-histidine codon with ahistidine codon and the light chain locus encodes an immunoglobulinlight chain comprising a variable domain of a reverse chimeric lightchain and an endogenous non-human light chain constant region (C_(L)).In a specific embodiment, the rearranged human V_(L)/J_(L) sequence isat the non-human kappa (K) locus, and the non-human C_(L) is a non-humanCκ. In one specific embodiment, the rearranged human variable regionsequence that comprises a substitution of at least one non-histidinecodon with a histidine codon is operably linked to the non-humanimmunoglobulin light chain constant region sequence, e.g., the non-humanCκ gene sequence. In one embodiment, the non-human immunoglobulin lightchain constant region sequence is an endogenous non-human sequence. Inone embodiment, the non-human animal is a mouse and the Cκ gene sequenceis a mouse Cκ gene sequence. In one embodiment, the C_(L) is a humanC_(L). In one embodiment, the rearranged human immunoglobulin lightchain variable region sequence comprising a substitution of at least onenon-histidine codon with a histidine codon is at the endogenousnon-human (e.g., mouse) immunoglobulin light chain locus (K locus).Exemplary embodiments of the locus are presented in FIGS. 8C, 8E, 14C,and 14D.

In one embodiment, the genetically modified non-human animal is a mouse,and the variable region locus of the mouse is a κ light chain locus, andthe κ light chain locus comprises a mouse κ intronic enhancer, a mouse κ3′ enhancer, or both an intronic enhancer and a 3′ enhancer.

In one embodiment, the non-human animal (e.g., a rodent, e.g., a rat ora mouse) comprises a nonfunctional immunoglobulin lambda (λ) light chainlocus. In a specific embodiment, the λ, light chain locus comprises adeletion of one or more sequences of the locus, wherein the one or moredeletions renders the λ, light chain locus incapable of rearranging toform a light chain gene. In another embodiment, all or substantially allof the V_(L) gene segments of the λ, light chain locus are deleted. Inone embodiment, the non-human animal (e.g., rodent, e.g. mouse or rat)comprises a rearranged human immunoglobulin light chain variable regionsequence comprising a substitution of at least one non-histidine codonwith a histidine codon, and lacks a functional unrearrangedimmunoglobulin light chain variable region, e.g., endogenousunrearranged light chain variable region. In one embodiment, therearranged, histidine-substituted human immunoglobulin light chainvariable region gene sequence replaces endogenous unrearrangedimmunoglobulin light chain variable region gene sequence. In anotherembodiment, the non-human animal (e.g., rodent, e.g., mouse or rat)comprises no more than two human V_(L) and one or more, e.g., two ormore, human J_(L) segments wherein each of the no more than two humanV_(L) and, optionally, one or more, e.g., two or more, human J_(L)segments comprise a substitution of at least one non-histidine codonwith a histidine codon, and wherein the animal lacks a functionalendogenous non-human light chain variable region; in one embodiment, thehistidine-substituted sequence replaces endogenous unrearrangedimmunoglobulin light chain variable region gene sequence. In anotherembodiment, the non-human animal (e.g., rodent, e.g., mouse or rat)comprises no more than two human V_(L) and one or more, e.g., two ormore, human J_(L) segments wherein each of the no more than two humanV_(L) and/or human J_(L) segments comprise a substitution of at leastone non-histidine codon with a histidine codon, and wherein the animallacks a functional endogenous non-human light chain variable region; inone embodiment, the histidine-substituted sequence replaces endogenousunrearranged immunoglobulin light chain variable region gene sequence.

In one embodiment, the animal makes a light chain that comprises asomatically mutated variable domain derived from a human variable regionsequence that comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the light chaincomprises a somatically mutated variable domain derived from a humanvariable region sequence that comprises a substitution of at least onenon-histidine codon with a histidine codon, and a non-human or human Cκregion. In one embodiment, the non-human animal does not express a λ,light chain.

One skilled in the art would appreciate that although substitution(s) ofat least one non-histidine residue with a histidine residue isgenetically engineered into the human immunoglobulin light chainvariable region, due to somatic hypermutations, not all antibodies thatare generated in the genetically modified non-human animal will harborthat histidine residue(s) at engineered position(s). However, generationof a wide repertoire of antibodies in the non-human animal will allow toselect for in vivo generated antigen-specific antibodies that displayhigh affinity for an antigen of interest while retaining histidinemodifications introduced into the germline and, preferably, exhibitingpH-dependent antigen binding.

Thus, in one embodiment, the animal retains at least one histidine aminoacid introduced by substitution of at least one non-histidine codon witha histidine codon in its variable region gene. In one embodiment, theanimal retains all or substantially all histidine substitutions in itssomatically mutated light chain variable domain that were introducedinto its variable region gene.

In one embodiment, the genetically modified non-human animal describedherein also comprises in its genome, e.g., in its germline, anunrearranged immunoglobulin heavy chain variable region comprisingV_(H), D_(H), and J_(H) gene segment sequences. In one embodiment, theV_(H), D_(H), and J_(H) gene segment sequences are human V_(H), D_(H),and J_(H) gene segment sequences, and the unrearranged immunoglobulinheavy chain variable region is a human heavy chain variable region. Inone embodiment, the human V_(H), D_(H), and J_(H) gene segment sequencesare operably linked to non-human heavy chain constant region sequence.In one embodiment, the non-human heavy chain constant region sequence isan endogenous non-human heavy chain constant region sequence. In anotherembodiment, the heavy chain constant region sequence is a human heavychain constant region sequence. In one embodiment, the human heavy chaingene segment sequences are at the endogenous non-human immunoglobulinheavy chain locus. In one embodiment, the human immunoglobulin heavychain variable region sequence comprised in a non-human animal alsocomprises a substitution of at least one non-histidine codon encoded bythe corresponding germline sequence for a histidine codon.

In one embodiment, the non-human animal described herein expresses animmunoglobulin light chain that comprises a non-human light chainconstant region sequence. In one embodiment, the non-human animalexpresses an immunoglobulin light chain that comprises a human lightchain constant region sequence.

In one embodiment, the non-human animal described herein expresses animmunoglobulin heavy chain that comprises a non-human sequence selectedfrom a C_(H)1 sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3sequence, and a combination thereof.

In one embodiment, the non-human animal expresses an immunoglobulinheavy chain that comprises a human sequence selected from a C_(H)1sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3 sequence, and acombination thereof.

In the embodiment where the animal comprises a single rearranged humanimmunoglobulin light chain variable region comprising a substitution ofat least one non-histidine codon with a histidine codon, or wherein theanimal comprises no more than two unrearranged human V_(L) gene segmentsand one or more, e.g., two or more (e.g., 2, 3, 4, or 5), unrearrangedhuman J_(L) gene segments wherein each unrearranged human V_(L) and,optionally, human J_(L) gene sequence(s), comprise substitution of atleast one non-histidine codon for a histidine codon (or wherein eachunrearranged human V_(L) and/or human J_(L) gene sequence comprisesubstitution of at least one non-histidine codon for a histidine codon),said variable region sequence or human V_(L) and J_(L) segments in thegermline of the animal are at an endogenous non-human immunoglobulinlight chain locus. In a specific embodiment, the rearrangedimmunoglobulin light chain sequence comprising a substitution of atleast one non-histidine codon with a histidine codon in the germline ofthe animal replaces all or substantially all endogenous non-human lightchain V and J segment sequences at the endogenous non-humanimmunoglobulin light chain locus. In another specific embodiment, the nomore than two unrearranged human V_(L) gene segments and one or more,e.g., two or more (e.g., 2, 3, 4, or 5), unrearranged human J_(L) genesegments wherein each unrearranged human V_(L) and, optionally, humanJ_(L) gene sequence(s), comprise substitution of at least onenon-histidine codon for a histidine codon (or wherein each unrearrangedhuman V_(L) and/or human J_(L) gene sequence comprise substitution of atleast one non-histidine codon for a histidine codon) in the germline ofthe animal replace all or substantially all endogenous non-human lightchain V and J segment sequences at the endogenous non-humanimmunoglobulin light chain locus.

In one embodiment, the non-human animal comprises a replacement ofendogenous V_(H) gene segments with one or more human V_(H) genesegments, wherein the human V_(H) gene segments are operably linked to anon-human C_(H) region gene, such that the non-human animal rearrangesthe human V_(H) gene segments and expresses a reverse chimericimmunoglobulin heavy chain that comprises a human V_(H) domain and anon-human C_(H). In one embodiment, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments.

A non-human animal, e.g., a mouse, comprising in its genome, e.g., inits germline, a limited repertoire of human immunoglobulin light chainvariable regions, e.g., a single rearranged human immunoglobulin lightchain variable region (e.g., Vκ1-39/Jκ5 or Vκ3-20/Jκ1), e.g., no morethan two unrearranged human V_(L) gene segments and one or more (e.g.,two or more) human J_(L) gene segments, with a substitution(s) of atleast one non-histidine codon with a histidine codon and a diverserepertoire of unrearranged human V_(H), D_(H), and J_(H) segments,described herein, is capable of generating antigen binding proteinsencoded by heavy chain variable region sequences derived from variouspermutations of unrearranged human V_(H), D_(H), and J_(H) segments,wherein the V_(H), D_(H), and J_(H) segments present in the heavy chainvariable sequences are derived from all or substantially all functionalhuman V_(H), D_(H), and J_(H) segments present in the genome of theanimal. Various available possibilities for heavy chain variable domainsequences expressed in the cells, e.g., B cells, of the geneticallymodified animals described herein (i.e., derived from combinations ofvarious functional human V, D, and J segments) are described in U.S.Application Publication Nos. 2011/0195454, 2012/0021409, 2012/0192300and 2013/0045492, 2013/0185821, and 2013/0198880, all incorporatedherein by reference. In various embodiments, the rearranged humanimmunoglobulin light chain variable region sequence or no more than twounrearranged human V_(L) gene segments and one or more, e.g., two ormore, human J_(L) gene segments comprising substitution(s) of at leastone non-histidine codon with a histidine codon described herein and theunrearranged human immunoglobulin heavy chain variable region sequenceare comprised in the germline of the non-human animal. In someembodiment, the non-human animals described herein are capable ofgeneration of epitope binding proteins encoded by their dual light chainlocus.

In one embodiment, the non-human animal comprising thehistidine-substituted single rearranged human immunoglobulin light chainvariable region sequences comprises one copy of one or both of thesingle rearranged human immunoglobulin light chain variable regionsequence comprising substitution(s) of at least one non-histidine codonwith a histidine codon and the unrearranged human immunoglobulin heavychain variable region sequence. In another embodiment, the non-humananimal comprises two copies of one or both of the rearranged humanimmunoglobulin light chain variable region sequence comprisingsubstitution(s) of at least one non-histidine codon with a histidinecodon and the unrearranged human immunoglobulin heavy chain variableregion sequence. Thus, the non-human animal may be homozygous orheterozygous for one or both the rearranged human immunoglobulin lightchain variable region sequence comprising substitution(s) of at leastone non-histidine codon with a histidine codon and the unrearrangedhuman immunoglobulin heavy chain variable region sequence.

In another embodiment, the non-human animal comprising no more than twohistidine-substituted human V_(L) gene segments and one or a pluralityof J_(L) segments (e.g., two or more J_(L) segments) is such that thetwo histidine-substituted human V_(L) gene segments are juxtaposed inthe genome of the animal. In some embodiments, the non-human animalcomprises one copy of the locus wherein the two histidine-substitutedhuman V_(L) gene segments are juxtaposed; in other embodiments, thenon-human animal comprises two copies of the locus wherein the twohistidine-substituted human V_(L) gene segments are juxtaposed. Thus, insome embodiments, the non-human animal is either homozygous orheterozygous for an immunoglobulin light chain locus comprising no morethat two juxtaposed human V_(L) gene segments. In some embodiments, thetwo histidine-substituted human V_(L) gene segments are at differentloci (e.g., a heterozygote, comprising a first histidine-substitutedhuman V_(L) segment at a first light chain allele, and a secondhistidine-substituted human V_(L) segment at a second light chainallele, wherein the first and the second human V_(L) segments are notidentical) in the genome of the animal. In some embodiments, the animalis also heterozygous or homozygous for unrearranged human immunoglobulinheavy chain variable locus. In some embodiments, the two humanhistidine-substituted V_(L) gene segments are a human Vκ1-39 genesegment and a human Vκ3-20 gene segment. In one embodiment, the humanJ_(L) gene segment is selected from the group consisting of Jκ1, Jκ2,Jκ3, Jκ4, Jκ5, and pairwise combinations thereof. In variousembodiments, a provided genetically engineered non-human animal isincapable of expressing an immunoglobulin light chain that contains anendogenous V_(L) gene segment. For example, in some embodiments, aprovided genetically engineered non-human animal contains a geneticmodification that inactivates and/or removes part or all of anendogenous V_(L) gene segment.

In addition to genetically modified non-human animals comprising intheir genome an immunoglobulin light chain variable region gene sequence(e.g., an immunoglobulin light chain variable region sequence with alimited repertoire of light chain variable gene segments, e.g., a singlerearranged immunoglobulin light chain variable region gene sequence,e.g., a sequence comprising no more than two V_(L) gene segments and oneor a plurality of J_(L) gene segments) comprising substitution(s) of atleast one non-histidine codon with a histidine codon (e.g., in CDR3 ofthe light chain), also provided herein are genetically modifiednon-human animals comprising an immunoglobulin light chain variableregion gene sequence with one or more additions/insertions of histidinecodon(s), such that the expressed variable domain comprises anadditional amino acid(s) which, if not subject to somatic hypermutation,is a histidine. In one embodiment, such additions of histidine codonscan be introduced by inserting human histidine-substituted D_(H)sequence into the human light chain locus of the mouse. Also, theanimals described herein comprising histidine modifications in theirlight chain variable domains may also contain histidine modifications intheir heavy chain variable domains, e.g., animals may also containhistidine modifications in the human heavy chain variable domains.

The genetically modified non-human animal comprising a humanimmunoglobulin light chain variable region gene sequence with asubstitution of at least one non-histidine codon with a histidine codondescribed herein may be selected from a group consisting of a mouse,rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat,chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). Forthe non-human animals where suitable genetically modifiable ES cells arenot readily available, methods distinct from those described herein areemployed to make a non-human animal comprising the genetic modification.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo. In anotherembodiment, a non-human animal described herein may be generated viatetraploid complementation.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the genetically modified non-human animal is arodent. In one embodiment, the genetically modified non-human animal isa rat or a mouse. In one embodiment, the animal is a mouse. Thus, in oneembodiment, provided herein is a genetically modified mouse comprisingin its genome, e.g., in its germline, a single rearranged humanimmunoglobulin light chain variable region comprising human V_(L) andJ_(L) gene sequences, wherein the single rearranged human immunoglobulinlight chain variable region comprises a substitution of at leastnon-histidine codon encoded by the corresponding human germline sequencewith a histidine codon. In one embodiment, the mouse lacks a functionalunrearranged immunoglobulin light chain variable region (e.g., lacksfunctional unrearranged V and J gene segment sequences). In oneembodiment, the rearranged human immunoglobulin light chain variableregion with histidine codon substitution(s) is Vκ1-39/Jκ or Vκ3-20/Jκvariable region. In one embodiment the J segment sequence is selectedfrom Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In one embodiment the J segmentsequence is Jκ1 or Jκ5. In one embodiment, the substitution of at leastone non-histidine codon with a histidine codon is in the nucleotidesequence encoding a CDR3 region. In one embodiment, wherein therearranged variable region sequence is Vκ1-39/Jκ5 sequence, thehistidine substitution(s) is designed to express at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the rearranged variable region sequence isVκ3-20/Jκ1 sequence, the histidine substitution(s) is designed toexpress at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, the rearranged immunoglobulinlight chain variable region with substituted histidine codon(s) isoperably linked to an endogenous mouse immunoglobulin constant regiongene sequence (e.g., Cκ gene sequence). In one embodiment, the mousefurther comprises in its genome, e.g., in its germline, an unrearrangedimmunoglobulin heavy chain variable region comprising human V_(H),D_(H), and J_(H) segments. In one embodiment, human V_(H), D_(H), andJ_(H) segments are operably linked to an endogenous mouse immunoglobulinheavy chain constant region gene sequence. In various embodiments, therearranged human immunoglobulin light chain variable region sequencecomprising substitution(s) of at least one non-histidine codon with ahistidine codon and the unrearranged human immunoglobulin heavy chainvariable region sequence are comprised in the germline of the mouse.

Also, in some embodiments, provided herein is a genetically modifiedmouse that comprises in its genome, e.g., in its germline, a limitedrepertoire, e.g., no more than two, unrearranged human V_(L) genesegments and one or more, e.g., two or more (e.g., 2, 3, 4, or 5),unrearranged human J_(L) gene segments wherein each unrearranged humanV_(L) and/or human J_(L) gene segment comprises substitution of at leastone non-histidine codon for a histidine codon, e.g., at least onenon-histidine codon present in the corresponding human germline sequencefor a histidine codon. In some embodiments, provided herein is agenetically modified mouse that comprises in its genome, e.g., in itsgermline, a limited repertoire, e.g., no more than two, unrearrangedhuman V_(L) gene segments and one or more, e.g., two or more (e.g., 2,3, 4, or 5), unrearranged human J_(L) gene segments wherein eachunrearranged human V_(L) and, optionally, human J_(L) gene segment(s)comprise substitution of at least one non-histidine codon for ahistidine codon, e.g., at least one non-histidine codon present in thecorresponding human germline sequence for a histidine codon. In oneembodiment, the mouse lacks a functional unrearranged endogenous mouseimmunoglobulin light chain variable region (e.g., lacks functionalunrearranged endogenous immunoglobulin V and J gene segment sequences).In one embodiment, the no more than two unrearranged human V_(L) genesegments are Vκ1-39 and Vκ3-20 gene segments. In one embodiment the Jsegment sequence is selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, andcombinations thereof. In one embodiment, the substitution of at leastone non-histidine codon with a histidine codon is in the nucleotidesequence encoding a CDR3 region. In one embodiment, wherein theunrearranged V_(L) segment is a Vκ1-39 gene segment, the histidinesubstitution(s) is designed to express at a position selected from 105,106, 108, 111, and a combination thereof. In another embodiment, whereinthe unrearranged V_(L) segment is a Vκ3-20 segment, the histidinesubstitution(s) is designed to express at a position selected from 105,106, 107, 109, and a combination thereof. In one embodiment,immunoglobulin light chain variable region sequence comprising no morethan two human V_(L) gene segments and one or more, e.g., two or more,human J_(L) gene segments, comprising substitution(s) of histidinecodon(s), is operably linked to an endogenous mouse immunoglobulinconstant region gene sequence (e.g., Cκ gene sequence). In oneembodiment, immunoglobulin light chain variable region sequencecomprising no more than two human V_(L) gene segments and one or more,e.g., two or more, human J_(L) gene segments, comprising substitution(s)of histidine codon(s), is operably linked to a human immunoglobulinconstant region gene sequence (e.g., Cκ gene sequence). In oneembodiment, the mouse further comprises in its genome, e.g., in itsgermline, an unrearranged immunoglobulin heavy chain variable regioncomprising human V_(H), D_(H), and J_(H) segments. In one embodiment,human V_(H), D_(H), and J_(H) segments are operably linked to anendogenous mouse immunoglobulin heavy chain constant region genesequence. In another embodiment, human V_(H), D_(H), and J_(H) segmentsare operably linked to a human immunoglobulin heavy chain constantregion sequence. In various embodiments, the immunoglobulin light chainvariable region sequence comprising no more than two human V_(L) genesegments and one or more, e.g., two or more, human J_(L) gene segments,comprising substitution(s) of histidine codon(s), and the unrearrangedhuman immunoglobulin heavy chain variable region sequence are comprisedin the germline of the mouse.

Also provided herein are targeting vectors for generating geneticallymodified non-human animals, e.g., mice, described herein. In one aspect,provided is a targeting vector comprising, d of the vector: a 5′ mousehomology arm, a human or mouse immunoglobulin promoter, a human or mouseleader sequence, a human variable region selected from a rearrangedhuman Vκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1 and comprising asubstitution of at least one non-histidine codon with a histidine codon,and a 3′ mouse homology arm. In one embodiment, the 5′ and 3′ homologyarms target the vector to a sequence 5′ with respect to an enhancersequence that is present 5′ and proximal to the mouse Cκ gene. Inanother embodiment, the targeting vector comprises a 5′ mouse homologyarm followed by a selection cassette flanked by recombination sites,human or mouse immunoglobulin promoter, human or mouse leader sequence,a human variable region selected from a rearranged human Vκ1-39Jκ5 or arearranged human Vκ3-20Jκ1 and comprising a substitution of at least onenon-histidine codon with a histidine codon, followed by the 3′ mousehomology arm that comprises mouse enhancers and constant region (Cκ)sequences.

In another aspect, provided herein is a targeting vector comprising: a5′ mouse homology arm; a human variable region comprising no more thantwo unrearranged human V_(L) gene segments and one or a plurality (e.g.,two or more, e.g., 2, 3, 4, or 5) of unrearranged human J_(L) genesegments wherein each unrearranged human V_(L) and, optionally, humanJ_(L) gene segment sequence(s), comprise substitution of at least onenon-histidine codon for a histidine codon, e.g., at least onenon-histidine codon present in the human germline sequence for ahistidine codon, and wherein each unrearranged V_(L) gene segment isoperably linked to a human or a mouse leader sequence and a human or amouse promoter; and a 3′ mouse homology arm. In one embodiment, the 5′and 3′ homology arms target the vector to a sequence 5′ with respect toan enhancer sequence that is present 5′ and proximal to the mouse Cκgene. In another embodiment, the targeting vector comprises: a 5′ mousehomology arm followed by a selection cassette flanked by recombinationsites; a human variable region comprising no more than two unrearrangedhuman V_(L) gene segments and one or a plurality (e.g., two or more,e.g., 2, 3, 4, or 5) of unrearranged human J_(L) gene segments whereineach unrearranged human V_(L) and, optionally, human J_(L) gene segmentsequence(s) comprise substitution of at least one non-histidine codonfor a histidine codon, e.g., at least one non-histidine codon present inthe germline sequence for a histidine codon, and wherein eachunrearranged V_(L) gene segment is operably linked to a human or a mouseleader sequence and a human or a mouse promoter; followed by the 3′mouse homology arm that comprises mouse enhancers and constant region(Cκ) sequences.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., bacterial cells, EScells, etc.) that have integrated the construct of interest. A number ofsuitable selection cassettes are known in the art. Commonly, a selectioncassette enables positive selection in the presence of a particularantibiotic (e.g., Neo, Hyg, Pur, CM, Spec, etc.). In addition, aselection cassette may be flanked by recombination sites, which allowdeletion of the selection cassette upon treatment with recombinaseenzymes. Commonly used recombination sites are loxP and Frt, recognizedby Cre and Flp enzymes, respectively, but others are known in the art.

In one embodiment, the promoter is a human immunoglobulin variableregion gene segment promoter. In a specific embodiment, the promoter isa human Vκ3-15 promoter. In another embodiment, the promoter is a humanVκ1-39 of Vκ3-20 promoter. In one embodiment, the leader sequence is amouse leader sequence. In a specific embodiment, the mouse leadersequence is a mouse Vκ3-7 leader sequence. In another embodiment, theleader sequence is a human leader sequence. In a specific embodiment,the human leader sequence is a human Vκ1-39 or Vκ3-20 leader sequence.Exemplary embodiments of the targeting vectors comprising a singlerearranged human variable region are presented in FIGS. 8B and 14B.Exemplary embodiments of the targeting vectors comprising a humanvariable region comprising no more than two unrearranged human V_(L)gene segments and a plurality of human J_(L) gene segments is presentedin FIGS. 31A and 33A.

In one aspect, a targeting vector is provided as described above, but inplace of the 5′ mouse homology arm the human or mouse promoter isflanked 5′ with a site-specific recombinase recognition site (SRRS), andin place of the 3′ mouse homology arm the human V_(L) region is flanked3′ with an SRRS.

Also provided herein are methods of making genetically modifiednon-human animals (e.g., rodents, e.g., mice or rats) described herein.In one aspect, the method for making a genetically modified non-humananimal described herein utilizes a targeting vector, made usingVELOCIGENE® technology, introducing the construct into ES cells, andintroducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. Histidinemodifications may be introduced into the targeting vector using avariety of molecular biology techniques, e.g., site directed mutagenesisor de novo DNA synthesis. Upon completion of gene targeting, ES cells ofgenetically modified non-human animals are screened to confirmsuccessful incorporation of exogenous nucleotide sequence of interest orexpression of exogenous polypeptide. Numerous techniques are known tothose skilled in the art, and include (but are not limited to) Southernblotting, long PCR, quantitative PCR (e.g., real-time PCR usingTAQMAN®), fluorescence in situ hybridization, Northern blotting, flowcytometry, Western analysis, immunocytochemistry, immunohistochemistry,etc. In one example, non-human animals (e.g., mice) bearing the geneticmodification of interest can be identified by screening for loss ofmouse allele and/or gain of human allele using a modification of alleleassay described in Valenzuela et al. (2003) High-throughput engineeringof the mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659. Other assays that identify a specificnucleotide or amino acid sequence in the genetically modified animalsare known to those skilled in the art.

Thus, in one embodiment, the method of generating genetically modifiednon-human animals comprises replacing an immunoglobulin light chainvariable region gene sequence in the animal with a human immunoglobulinlight chain variable region gene sequence (comprising human V_(L) andJ_(L) gene segments) wherein the human immunoglobulin variable regiongene sequence comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the substitution of atleast one non-histidine codon with a histidine codon is in thenucleotide sequence encoding a CDR region, e.g., a CDR3 region.

In one embodiment, the method of generating genetically modifiednon-human animals described herein comprises replacing an immunoglobulinlight chain variable region gene sequence in the animal with a singlerearranged human immunoglobulin light chain variable region genesequence comprising human V_(L) and J_(L) gene segment sequences,wherein the single rearranged human immunoglobulin variable region genesequence comprises at least one histidine that is not encoded by thecorresponding human germline sequence, e.g., wherein the singlerearranged human immunoglobulin variable region gene sequence comprisesa substitution of at least one non-histidine codon with a histidinecodon, e.g., at least one histidine codon encoded by the correspondinghuman germline sequence with a histidine codon. In one embodiment, thesubstitution is in a CDR codon. In one embodiment, the substitution isof one, two, three, four, or more CDR3 codon(s). In one embodiment, thesingle rearranged human immunoglobulin light chain variable region genesequence is based on the human germline rearranged light chain variableregion sequence selected from Vκ1-39Jκ5 and Vκ3-20Jκ1. Thus, in oneembodiment, where the single rearranged human immunoglobulin light chainvariable region gene sequence is derived from Vκ1-39Jκ5, replacement ofat least one non-histidine codon with histidine codon is designed toexpress a histidine at positions selected from 105, 106, 108, 111, and acombination thereof. In one embodiment, where the single rearrangedhuman immunoglobulin light chain variable region gene sequence isderived from Vκ3-20κ1, replacement of at least one non-histidine codonwith a histidine codon is designed to express a histidine at positionselected from 105, 106, 107, 109, and a combination thereof.

In yet another embodiment, the method of generating genetically modifiednon-human animals described herein comprises replacing an immunoglobulinlight chain variable region gene sequence in the animal with animmunoglobulin light chain variable gene sequence comprising no morethan two unrearranged human V_(L) gene segments and one or a plurality(e.g., two or more, e.g., 2, 3, 4, or 5) of unrearranged human J_(L)gene segments wherein each unrearranged human V_(L) and, optionally,human J_(L) gene sequence(s) comprise at least one histidine that is notencoded by the corresponding human germline variable gene segments,e.g., wherein each unrearranged human V_(L) and, optionally, human J_(L)gene sequence(s) comprise a substitution of at least one non-histidinecodon for a histidine codon, e.g., at least one non-histidine codonpresent in the human germline sequence for a histidine codon. In oneembodiment, the substitution is in a CDR codon. In one embodiment, thesubstitution is of one, two, three, four, or more CDR3 codon(s). In oneembodiment, the no more than two unrearranged human V_(L) gene segmentsare Vκ1-39 and Vκ3-20 gene segments. In one embodiment, the unrearrangedhuman J_(L) segments are selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, andcombinations thereof. Thus, in one embodiment, wherein the unrearrangedhuman V_(L) gene segment is Vκ1-39, replacement of at least onenon-histidine codon with histidine codon is designed to express ahistidine at positions selected from 105, 106, 108, 111, and acombination thereof. In one embodiment, wherein the unrearranged humanV_(L) gene segment is Vκ3-20, replacement of at least one non-histidinecodon with a histidine codon is designed to express a histidine atposition selected from 105, 106, 107, 109, and a combination thereof.

In another embodiment, the method of generating a non-human animaldescribed herein (i.e., comprising a genetically modified immunoglobulinlight chain locus described herein) comprises modifying a genome of anon-human animal to delete or render non-functional endogenousimmunoglobulin light chain V and J segments in an immunoglobulin lightchain locus, and placing in the genome (1) a single rearranged humanlight chain variable region gene sequence comprising a substitution ofat least one non-histidine codon with a histidine codon or (2) animmunoglobulin light chain variable gene sequence comprising no morethan two unrearranged human V_(L) gene segments and one or a plurality(e.g., two or more, e.g., 2, 3, 4, or 5) of unrearranged human J_(L)gene segments wherein each unrearranged human V_(L) and, optionally,human J_(L) gene sequence(s) comprise substitution of at least onenon-histidine codon for a histidine codon (or wherein each human V_(L)and/or human J_(L) gene sequence(s) comprise substitution of at leastone non-histidine codon for a histidine codon). In one embodiment, themethod results in a genetically modified non-human animal that comprisesa population of B cells enriched for antibodies exhibiting pH dependentbinding to an antigen of interest.

In some embodiments, the methods of generating genetically modifiednon-human animals described herein comprise replacing an immunoglobulinlight chain variable region gene sequence with human immunoglobulinlight chain variable gene region sequence comprising substitution(s) ofat least one non-histidine codon with a histidine codon in the animalthat also comprises a replacement of endogenous non-human immunoglobulinheavy chain variable region gene sequence with a human immunoglobulinheavy chain variable region gene sequence comprising at least one ofeach or a repertoire of human V_(H), D_(H), and J_(H) sequences, asdescribed above. In one embodiment, in order to generate a non-humananimal comprising a replacement of endogenous immunoglobulin light chainvariable region gene sequence human light chain variable region genesequence comprising a substitution of at least one non-histidine codonwith a histidine codon and a replacement of endogenous non-humanimmunoglobulin heavy chain variable region gene sequence with a humanimmunoglobulin heavy chain variable region gene sequence, the animalwith replacement of light chain variable region gene sequence is bred toan animal with replacement of heavy chain variable region gene sequence.

Inventors presently provide genetically engineered non-human animals(e.g., rodents, e.g., rats or mice) that express antigen-bindingproteins, e.g., antibodies, that comprise a universal light chain, e.g.,a human universal light chain (e.g., a light chain derived from a singlerearranged human immunoglobulin light chain variable region), or a lightchain with limited or restricted variable segment repertoire (e.g.,comprising no more than two unrearranged human V_(L) gene segments andone or a plurality (e.g., two or more) unrearranged human J_(L) genesegments) that comprises one or more histidine modifications, whereinthe antigen-binding proteins exhibit a pH-dependent antigen binding of atarget antigen. In some embodiments, the animals are geneticallyengineered to include a light chain CDR3 that comprises one or morehistidine modifications. In various embodiments, the light chain CDR3comprises two, three, or four or more histidine residues in a cluster.

In one embodiment, provided herein is a genetically engineered non-humananimal (e.g., a mouse or a rat) that comprises a population of B cellscharacterized by enhanced presence of histidines in immunoglobulin lightchains, e.g., immunoglobulin variable domains, e.g., immunoglobulinCDRs, compared to a wild type animal. In one embodiment, enhancement ofhistidine presence is about 2 to 4 fold. In one embodiment, enhancementof histidines is about 2 to 10 fold.

In one embodiment, provided herein is a genetically engineered non-humananimal that comprises a population of antigen-specific antibodies thatexpress histidine residue(s) as a result of codon modifications in thelight chain variable region gene sequence, and display pH-dependentbinding of target antigen. In one embodiment, these animals comprise apopulation of B cells that are enriched for antibodies, e.g.,antigen-specific antibodies, that display pH-dependent bindingproperties (e.g., decreased dissociative half-life (t_(1/2)), at acidicpH vs neutral pH) as compared to a population of antigen-specificantibodies generated in animals that do not comprise a substitution ofat least one non-histidine codon encoded by human germline sequence witha histidine codon in immunoglobulin light chain variable regiondescribed herein. In one embodiment, the enrichment of antigen-specificantibodies displaying pH-dependent antigen binding properties generatedin the genetically engineered animals described herein as compared tosimilar animals that do comprise histidine substitutions in light chainvariable region is greater than about 2 fold, e.g., greater than about 5fold, e.g., greater than about 10 fold. In one embodiment, theenrichment is about 2-3 fold. Thus, the genetically modified animals ofthe invention are enriched for antibodies with improved antibodyrecycling properties, which is desired in order to reducetarget-mediated clearance as well as to reduce the dose and/or dosingfrequency of a therapeutic antigen-binding protein developed based onsuch in vivo generated antibody format.

Thus, provided herein is an antigen-binding protein, generated ingenetically modified non-human animals described herein, wherein theantigen-binding protein displays pH-dependent antigen binding. In oneembodiment, the antigen-binding protein is an antibody, e.g.,antigen-specific antibody. In one embodiment, the antibody comprises alight chain which comprises a human light chain variable domain derivedfrom a rearrangement of human immunoglobulin light chain variable genesegments where at least one non-histidine codon was substituted for ahistidine codon in the germline gene sequence, and wherein the antibodyretains at least one histidine substitution in its expressed human lightchain variable domain. In another embodiment, the antibody comprises alight chain which comprises a human light chain variable domain derivedfrom a single rearranged human light chain variable region genesequence, wherein the single rearranged light chain variable region genesequence comprises a substitution of at least one non-histidine codonwith a histidine codon, and wherein the antibody retains at least onehistidine substitution in its expressed light chain variable domain. Inone embodiment, the antibody comprises a light chain derived from ahuman Vκ1-39/J or Vκ3-20/J (e.g., Vκ1-39Jκ5 or Vκ3-20Jκ1) rearrangement,wherein the human Vκ1-39J or Vκ3-20J gene sequence comprises asubstitution of at least one non-histidine codon with a histidine codon,and wherein the antibody retains at least one histidine substitution inits expressed light chain variable domain. In another embodiment, theantibody comprises a light chain which comprises a human light chainvariable domain derived from a rearrangement of light chain variableregion gene sequence present at the germline locus, wherein light chainvariable region gene sequence present at the germline locus comprises nomore than two unrearranged human V_(L) gene segments and one or aplurality (e.g., two or more) unrearranged human J_(L) gene segments andeach unrearranged human V_(L) gene segment and, optionally, human J_(L)gene segment(s), comprise substitution of at least one non-histidinecodon for a histidine codon, and wherein the antibody retains at leastone histidine substitution in its expressed light chain variable domain.In one embodiment, the antibody comprises a light chain derived from ahuman Vκ1-39 or Vκ3-20 rearranged with a J segment, wherein suchrearranged human Vκ1-39Jκ or Vκ3-20Jκ gene sequence comprises asubstitution of at least one non-histidine codon with a histidine codon,and wherein the antibody retains at least one histidine substitution inits expressed light chain variable domain. In some embodiments, theantibody retains all or substantially all histidine substitutions in itsexpressed light chain variable domain. In one embodiment, the antibodyretains at least 50%, at least at least 66%, at least 90%, at least 95%,at least 97%, at least 98%, at least 99% of all histidine substitutionsin its light chain variable domain. In one embodiment, the substitutionis of three non-histidine codons with three histidine codons in thenucleotide sequence encoding CDR3 of the light chain variable regiongene sequence, and the antibody retains all three histidinesubstitutions in its expressed light chain variable domain. In anotherembodiment, the substitution is of three non-histidine codons with threehistidine codons in the nucleotide sequence encoding CDR3 of the lightchain variable region gene sequence, and the antibody retains two orthree histidine substitutions in its expressed light chain variabledomain. In one embodiment, the substitution is of four non-histidinecodons with four histidine codons in the nucleotide sequence encodingCDR3 of the light chain variable region gene sequence, and the antibodyretains three or four histidine substitutions in its expressed lightchain variable domain. In other embodiments, the antibody retains one,two, three, four, and up to all histidine modifications in its expressedlight chain variable domain.

In one embodiment, the light chain of the antibody further comprises anon-human light chain constant region amino acid sequence, e.g.,endogenous light chain constant region amino acid sequence. In addition,the antibody, e.g., antigen-specific antibody, generated in agenetically modified non-human animal described herein also comprises aheavy chain which comprises a human heavy chain variable domain derivedfrom a rearrangement of human heavy chain V, D, and J segments. Humanheavy chain V, D, and J segments may be selected from a repertoire ofhuman heavy chain segments present at the endogenous non-human heavychain locus, e.g., at least one functional V, at least one functional D,and at least one functional J segment, e.g., up to a complete repertoireof functional human V, D, and J segments. Exemplary possiblerearrangements of human heavy chain variable segments may be gleanedfrom a listing of functional human V, D, and J segments in IMGTdatabase, and from U.S. Application Publication Nos. 2011/0195454,2012/0021409, 2012/0192309, and 2013/0045492, incorporated herein byreference. Furthermore, in one embodiment, the heavy chain of theantibody comprises a non-human heavy chain constant region amino acidsequence, e.g., an endogenous non-human heavy chain constant regionamino acid sequence. In one embodiment, the non-human heavy chainconstant region comprises C_(H)1, hinge, C_(H)2, and C_(H)3 domains. Inone embodiment, the antibody is an IgG, IgE, IgD, IgM, or IgA isotype.

Thus, in one embodiment, provided herein is a binding protein generatedin the genetically modified non-human animals described herein, whereinthe binding protein comprises a reverse chimeric light chain comprising(a) a light chain variable domain derived from a human Vκ1-39 to Jκrearrangement (e.g., Vκ1-39Jκ5 rearrangement) comprising a substitutionof at least one non-histidine codon with a histidine codon, wherein thelight chain retains at least one histidine substitution in its expressedlight chain variable domain and (b) a non-human, e.g., a mouse, lightchain constant region amino acid sequence, wherein the light chain isassociated with a reverse chimeric heavy chain comprising (a) a heavychain variable domain derived from a rearrangement of human V, D, and Jsegments, wherein the V, D, and J segments are selected from arepertoire of human V, D, and J segments present in the animal, and (b)a non-human, e.g., mouse, heavy chain constant region amino acidsequence. In one embodiment, the repertoire of human V, D, and Jsegments comprises at least one functional V, at least one functional D,and at least one functional J segment, e.g., up to a complete repertoireof functional human V, D, and J segments. In one embodiment, the heavyand the light chain constant domains are endogenous heavy and lightchain constant domains. In one embodiment, the heavy and light chainvariable domains are somatically mutated domains. In one embodiment, thesomatically mutated light chain domain retains at least one histidinesubstitution introduced into the germline sequence. In some embodiments,the somatically mutated light chain domain retains all or substantiallyall histidine substitutions introduced into the germline sequence. Inone embodiment, the antigen-binding protein displays pH-dependentantigen binding properties.

In another embodiment, provided herein is a binding protein generated inthe genetically modified non-human animals described herein, wherein thebinding protein comprises a reverse chimeric light chain comprising (a)a light chain variable domain derived from a human Vκ3-20 to Jκrearrangement (e.g., Vκ3-20Jκ1 rearrangement) comprising a substitutionof at least one non-histidine codon with a histidine codon, wherein thelight chain retains at least one histidine substitution in its expressedlight chain variable domain and (b) a non-human, e.g., a mouse, lightchain constant region amino acid sequence, wherein the light chain isassociated with a reverse chimeric heavy chain comprising (a) a heavychain variable domain derived from a rearrangement of human V, D, and Jsegments, wherein the V, D, and J segments are selected from arepertoire of human V, D, and J segments present in the animal, and (b)a non-human, e.g., mouse, heavy chain constant region amino acidsequence. In one embodiment, the repertoire of human V, D, and Jsegments comprises at least one functional V, at least one functional D,and at least one functional J segment, e.g., up to a complete repertoireof functional human V, D, and J segments. In one embodiment, the heavyand the light chain constant regions are endogenous heavy and lightchain constant regions. In one embodiment, the heavy and light chainvariable domains are somatically mutated domains. In one embodiment, thesomatically mutated light chain domain retains at least one histidinesubstitution introduced into the germline sequence. In some embodiments,the somatically mutated light chain domain retains all or substantiallyall histidine substitutions introduced into the germline sequence. Inone embodiment, the antigen-binding protein displays pH-dependentantigen binding properties.

In one embodiment, also provided herein is a B cell of the geneticallymodified animal described herein, that comprises in its germline ahistidine-modified human light chain variable region sequence, e.g., ahistidine-modified single rearranged human light chain variable regionsequence or a histidine-modified human light chain variable regionsequence comprising no more than two unrearranged human V_(L) genesegments and one or a plurality (e.g., two or more) unrearranged humanJ_(L) gene segments, described herein, and expresses an antigen-bindingprotein described herein. In one embodiment, the antigen-bindingprotein, e.g., an antibody, expressed in the B cell retains at least onehistidine residue introduced into the germline, and displayspH-dependent antigen-binding properties. In some embodiments, theantigen-binding protein, e.g., an antibody, expressed in the B cellretains all or substantially all histidine residues introduced into thegermline, and displays pH-dependent antigen-binding properties.

In various embodiments, the genetically modified non-human animaldescribed herein comprises a human light chain variable region genesequence, e.g., a histidine-modified single rearranged human light chainvariable region gene sequence (e.g., Vκ1-39Jκ5 or Vκ3-20Jκ1 sequence) ora histidine-modified human light chain variable region sequencecomprising no more than two unrearranged human V_(L) gene segments andone or a plurality (e.g., two or more) unrearranged human J_(L) genesegments, that comprises a substitution of at least one non-histidinecodon with a histidine codon (or an addition of a histidine codon intothe germline sequence). These additions or substitutions result in anon-human animal that comprises a population of B cells enriched forantigen-binding proteins with pH dependent binding properties for theirantigens. In one embodiment, antigen-binding proteins, e.g., antibodies,generated in the non-human animals described herein in response toantigen stimulation display pH dependent antigen binding whileexhibiting high affinity for the antigen at neutral pH, e.g., pH betweenabout 7.0 and about 8.0, e.g., pH between about 7.0 and about 7.4, e.g.,between about 7.2 and about 7.4, e.g., physiological pH. In oneembodiment, the affinity of the antigen-binding protein to its antigen,expressed as a dissociation constant (K_(D)) at a neutral pH is lessthan 10⁻⁶ M, e.g., less than 10⁻⁸ M, e.g., less than 10⁻⁹ M, e.g., lessthan 10⁻¹⁰ M, e.g., less than 10⁻¹¹ M, e.g., less than 10⁻¹² M.

In one embodiment, an antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animal described herein,exhibits reduced binding to its antigen in acidic pH (e.g., pH of 6.0 orlower, e.g., pH between about 5.0 and about 6.0, pH between about 5.75and about 6.0, e.g., pH of endosomal or lysosomal compartments) ascompared to neutral pH. In one embodiment, the antigen-binding protein,e.g., the antibody, generated in the genetically modified non-humananimal described herein, exhibits no binding to the antigen in acidicpH, while retaining binding to the antigen at neutral pH. In oneembodiment, an antigen-binding protein generated by the geneticallymodified non-human animal described herein, has a decrease indissociative half-life (t_(1/2)) at an acidic pH as compared to thedissociative half-life (t_(1/2)) of the antigen-binding protein at aneutral pH of at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, or atleast about 30-fold. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein expressed by the geneticallymodified non-human animal described herein has a t_(1/2) at an acidic pHand 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein expressed by the genetically modified non-humananimal described herein has a t_(1/2) at an acidic pH and 25° C. ofabout 2 min or less. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 25° C. of less than about 1 min.

Kinetic parameters, such as equilibrium dissociation constants (K_(D))and dissociative half-lives (t_(1/2)) can be calculated from kineticrate constant as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=ln2/(60*k_(d)).

In one embodiment, the antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animals describedherein, exhibits increased binding to FcRn molecule. As described above,FcRn is a receptor present inside the endosomal compartment that iscapable of binding immunoglobulins at an acidic pH and recycling themback to the surface. Screening antibody molecules in the geneticallymodified non-human animals described herein presents a uniqueopportunity to select for antibodies with three beneficial parameters:high affinity for an antigen, pH-dependent antigen binding (with weakerantigen binding at acidic pH) and increased binding to FcRn.

In one embodiment, a genetically modified non-human animal describedherein comprises a population of B cells in response to an antigen thatproduces and is enriched for antigen-binding proteins, e.g., antibodies,that, when reformatted into therapeutics, exhibit increased serum halflife upon administration of a therapeutic dose to a subject over anequivalent B cell population produced in response to the same antigen innon-human animals that do not comprise histidine modification(s) intheir human light chain variable region gene sequences. Thus, in oneembodiment, an antigen-binding protein, e.g., an antibody, produced inresponse to an antigen of interest in a genetically modified non-humananimal described herein, when reformatted into a therapeutic, exhibitsincreased serum half life upon administration of a therapeutic dose to asubject over a serum half life of an antigen-binding protein (whenreformatted into a therapeutic and administered at the same therapeuticdose) that was produced in response to the same antigen in a non-humananimal that does not comprise histidine modification(s) in its humanlight chain variable region gene sequence. In some embodiments, theincrease in serum half life is about 2 fold, e.g., about 5 fold, e.g.,about 10 fold, e.g., about 15 fold, e.g., about 20 fold, or greater.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a non-human as described herein is provided. In a specificembodiment, the cell is an embryonic stem (ES) cell.

In one aspect, a tissue derived from a non-human animal as describedherein is provided. In one embodiment, the tissue is derived fromspleen, lymph node or bone marrow of a non-human animal as describedherein.

In one aspect, a nucleus derived from a non-human animal as describedherein is provided. In one embodiment, the nucleus is from a diploidcell that is not a B cell.

In one aspect, a non-human cell is provided that is isolated from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein. In one embodiment, the cell is an ES cell. In one embodiment,the cell is a lymphocyte. In one embodiment, the lymphocyte is a B cell.In one embodiment, the B cell expresses a chimeric heavy chaincomprising a variable domain derived from a human heavy chain genesegment; and a light chain derived from a rearranged human Vκ1-39/Jsequence with a substitution of at least one non-histidine codon in thegermline with histidine codon, rearranged human Vκ3-20/J sequence with asubstitution of at least one non-histidine codon in the germline withhistidine codon, or a combination thereof wherein the light chaincomprises a substitution of at least one amino acid encoded in thegermline for a histidine; wherein the heavy chain variable domain isfused to a non-human or a human heavy chain constant region and thelight chain variable domain is fused to a non-human or a human lightchain constant region. In another embodiment, the B cell expresses achimeric heavy chain comprising a variable domain derived from a humanheavy chain gene segment; and a light chain derived from a rearrangementof a human Vκ1-39 to human J sequence with a substitution of at leastone non-histidine codon in the germline with a histidine codon orderived from a rearrangement of a human Vκ3-20 to human J sequence witha substitution of at least one non-histidine codon in the germline witha histidine codon wherein the light chain comprises a substitution of atleast one amino acid encoded in the germline for histidine; wherein theheavy chain variable domain is fused to a non-human or a human heavychain constant region and the light chain variable domain is fused to anon-human or a human light chain constant region.

In one aspect, a hybridoma is provided, wherein the hybridoma is madewith a B cell of a non-human animal as described herein. In a specificembodiment, the B cell is from a mouse as described herein that has beenimmunized with an immunogen comprising an epitope of interest, and the Bcell expresses a binding protein that binds the epitope of interest, thebinding protein has a somatically mutated human variable heavy chaindomain and a mouse C_(H); and has a human variable light chain domainderived from (1) a rearranged human Vκ1-39Jκ5, (2) a rearrangement ofhuman Vκ1-39 to a human J, (3) a rearranged human Vκ3-20Jκ1, or (4) arearrangement of human Vκ3-20 to a human J, each bearing a substitutionof at least one non-histidine codon in the germline with a histidinecodon, and a mouse C_(L); wherein the human light chain domain comprisesa substitution of at least one amino acid encoded in the germline with ahistidine.

Also provided is a cell expressing an antigen-binding protein generatedin the non-human animals described herein. In one embodiment, the cellis selected from CHO, COS, 293, HeLa, and a retinal cell expressing aviral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a non-human embryo is provided, wherein the embryocomprises a donor ES cell that is derived from a non-human animal asdescribed herein.

The non-human animals described herein are useful to generate B cellsthat express antibodies having histidines in a CDR3. An animal thatplaces histidines in a CDR3 is useful for making antibodies in general,and in particular useful for developing antibodies that bind a targetwith sufficient affinity at or around a neutral pH, but that either donot bind or that bind weaker to the same target at an acidic pH.

The non-human animal is useful to generate variable regions ofantibodies that can be used to make, e.g., human therapeutic bindingproteins that bind their targets by human immunoglobulin variabledomains that comprise the histidines in a CDR3. The altered binding at alower pH will in some circumstances allow faster turnover because thetherapeutic will bind a target on a cell's surface, be internalized inan endosome, and more readily or more rapidly dissociate from the targetin the endosome, so that the therapeutic can be recycled to bind yetanother molecule of target (e.g., on another cell or the same cell). Insome circumstances, this will result in the ability to dose thetherapeutic at a lower dose, or dose the therapeutic less frequently.This is particularly useful where it is not desirable to dosefrequently, or to administer above a certain dosage, for safety ortoxicity reasons. As a result, the serum half-life of the antibodytherapeutic when administered to a subject will be increased.

The non-human animal, e.g., rodent, e.g., mouse or rat, is useful in amethod for increasing the number of B cells in an animal that exhibit anantibody variable region having a CDR3 with one or more histidines init. The non-human animal is useful for generating antibody sequencesthat will exhibit pH-dependent antigen binding. The non-human animal isuseful for generating a greater number of antibody sequences, resultingfrom a single immunization, wherein the antibodies will exhibit apH-dependent antigen binding.

Antigen-Binding Proteins and Methods of Generating the Same

In one aspect, also provided herein are methods for generating humanantigen-binding proteins, e.g., antibodies, which exhibit pH-dependentantigen binding, from the genetically modified non-human animalsdescribed herein with standard methods used in the art.

Several techniques for producing antibodies have been described. Forexample, in various embodiments chimeric antibodies are produced in miceas described herein. Antibodies can be isolated directly from B cells ofan immunized mouse (e.g., see U.S. 2007/0280945A1) and/or the B cells ofthe immunized mouse can be used to make hybridomas (Kohler and Milstein,1975, Nature 256:495-497). DNA encoding the antibodies (human heavyand/or light chains) from non-human animals as described herein isreadily isolated and sequenced using conventional techniques. Hybridomaand/or B cells derived from non-human animals as described herein serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof monoclonal antibodies in the recombinant host cells. The DNA also maybe modified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the non-humansequences. Thus, once nucleic acid sequences of antibodies with desiredcharacteristics, e.g., affinity, epitope, pH-dependent antigen binding,etc., are determined, the non-human constant region gene sequences arereplaced with a desired human constant region sequences to generate afully human antibody containing a non-IgM isotype, for example, IgG1,IgG2, IgG3 or IgG4.

Thus, in one embodiment provided herein is a method of generating anantibody that exhibits pH-dependent antigen binding propertiescomprising generating a non-human animal (e.g., a mouse) as describedherein, immunizing a mouse with an antigen of interest, allowing anon-human animal to mount an immune response to the antigen, andselecting in the non-human animal an antigen-specific antibody thatexhibits pH dependent antigen binding properties, e.g., weaker bindingto the antigen at an acidic than at neutral pH.

Also provided herein are methods of making multi-specific antigenbinding proteins, e.g., bispecific or trispecific antigen-bindingproteins. These are molecules capable of binding more than one epitopewith high affinity. Advantages of the invention include the ability toselect suitably high binding (e.g., affinity matured) heavy chainimmunoglobulin chains each of which will associate with a single lightchain. In addition, advantages of the invention include the ability togenerate a multi-specific, e.g., a bispecific or trispecific,antigen-binding protein that exhibits pH-dependent antigen binding.Various aspects of using bispecific antibodies described herein belowmay also be applicable to trispecific or other multispecific antibodies.

Because of the dual nature of bispecific antibodies (i.e., may bespecific for different epitopes of one polypeptide or may containantigen-binding domains specific for more than one target polypeptide,see, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244), they offer many useful advantages fortherapeutic application. For example, the bispecific antibodies can beused for redirected cytotoxicity (e.g., to kill tumor cells), as avaccine adjuvant, for delivering thrombolytic agents to clots, forconverting enzyme activated prodrugs at a target site (e.g., a tumor),for treating infectious diseases, targeting immune complexes to cellsurface receptors, or for delivering immunotoxins to tumor cells.

The bispecific antibodies described herein can also be used in severaltherapeutic and non-therapeutic and/or diagnostic assay methods, suchas, enzyme immunoassays, two-site immunoassays, in vitro or in vivoimmunodiagnosis of various diseases (e.g., cancer), competitive bindingassays, direct and indirect sandwich assays, and immunoprecipitationassays. Other uses for the bispecific antibodies will be apparent tothose skilled in the art.

Several techniques for making bispecific antibody fragments fromrecombinant cell culture have been reported. However, synthesis andexpression of bispecific binding proteins has been problematic, in partdue to issues associated with identifying a suitable light chain thatcan associate and express with two different heavy chains, and in partdue to isolation issues. In various embodiments, compositions andmethods described herein provide the advantage of full length bispecificantibodies that do not require special modification(s) to maintaintraditional immunoglobulin structure by increasing stability/interactionof the components. In various embodiments, such modification(s) hasproven cumbersome and served as an obstacle to development of bispecificantibody technology and their potential use in treating for humandisease. Thus, in various embodiments, through providing a naturalimmunoglobulin structure (i.e., full length) having the added propertyof multiple specificities, full length bispecific antibodies maintaintheir critical effector functions that previous bispecific fragmentslacked, and further provide therapeutics that demonstrate the importantpharmacokinetic parameter of a longer half-life.

Methods and compositions described herein allow for a geneticallymodified mouse to select, through otherwise natural processes, asuitable light chain that can associate and express with more than oneheavy chain, including heavy chains that are somatically mutated (e.g.,affinity matured), wherein the light chain further confers upon theantigen-binding protein its pH-dependent antigen binding property. Humanheavy and light chain variable region sequences from suitable B cells ofimmunized mice as described herein that express affinity maturedantibodies having reverse chimeric heavy chains (i.e., human variableand mouse constant) can be identified and cloned in frame in anexpression vector with a suitable human constant region gene sequence(e.g., a human IgG1). Two such constructs can be prepared, wherein eachconstruct encodes a human heavy chain variable domain that binds adifferent epitope. One of the human light chain variable regions (e.g.,human Vκ1-39/J or human Vκ3-20/J, e.g., Vκ1-39Jκ5 or human Vκ3-20Jκ1),comprising a substitution of at least one non-histidine codon with ahistidine codon, can be fused in frame to a suitable human light chainconstant region gene (e.g., a human κ constant gene). These three fullyhuman heavy and light constructs can be placed in a suitable cell forexpression. The cell will express two major species: a homodimeric heavychain with the identical light chain, and a heterodimeric heavy chainwith the identical light chain. To allow for a facile separation ofthese major species, one of the heavy chains is modified to omit aProtein A-binding determinant, resulting in a differential affinity of ahomodimeric binding protein from a heterodimeric binding protein.Compositions and methods that address this issue are described in U.S.Ser. No. 12/832,838, filed 25 Jun. 2010, entitled “Readily IsolatedBispecific Antibodies with Native Immunoglobulin Format,” published asUS 2010/0331527A1, hereby incorporated by reference. Once the speciecomprising heterodimeric heavy chain with an identical light chain isselected, this bi-specific antigen binding protein can be screened toconfirm the retention of its pH-dependent antigen binding property.

Alternatively, bispecific or trispecific antibodies can be preparedutilizing antigen-specific light chain derived from a mouse comprising adual light chain locus, e.g., a light chain locus that comprises no morethan two human V_(L)s and one or a plurality (e.g., two or more) humanJ_(L) gene segment sequences, and a limited repertoire of human heavychains (e.g., a single rearranged human heavy chain variable region).Such antigen-specific, histidine-modified, reverse chimeric (humanvariable mouse constant) light chain can be used to deriveantigen-specific light chain variable region sequence that can be clonedin frame into an expression vector with a suitable human light chainconstant region sequence. An antigen-specific human heavy chain variableregion(s) (specific for a different epitope on the same or differentantigen than the antigen-specific light chain) from a mouse comprising auniversal light chain locus, e.g., a light chain locus comprising asingle rearranged light chain variable region gene sequence, can becloned in frame into an expression vector comprising human heavy chainconstant region sequence, and the antigen-specific human light and heavychains can be co-expressed in a suitable cell to obtain a bispecific ortrispecific human antibody. Alternatively, a previously selectedantigen-specific heavy chain, e.g., a heavy chain from an antibody thatcomprises a light chain derived from the same variable region genesegment as the one used in the dual light chain mouse locus may becloned in frame into an expression vector comprising human heavy chainconstant region sequence, and the antigen-specific human light and heavychains can be co-expressed in a suitable cell to obtain a bispecific ortrispecific human antibody. In one embodiment, such antibody displayspH-dependent antigen binding, e.g., due to histidine substitutions inthe light chain.

In one aspect, an epitope-binding protein as described herein isprovided, wherein human light chain and heavy chain variable regionsequences are derived from animals described herein that have beenimmunized with an antigen comprising an epitope of interest.

In one embodiment, an epitope-binding protein is provided that comprisesa first and a second polypeptide, the first polypeptide comprising, fromN-terminal to C-terminal, a first epitope-binding region thatselectively binds a first epitope, followed by a constant region thatcomprises a first C_(H)3 region of a human IgG selected from IgG1, IgG2,IgG4, and a combination thereof; and, a second polypeptide comprising,from N-terminal to C-terminal, a second epitope-binding region thatselectively binds a second epitope, followed by a constant region thatcomprises a second C_(H)3 region of a human IgG selected from IgG1,IgG2, IgG4, and a combination thereof, wherein the second C_(H)3 regioncomprises a modification that reduces or eliminates binding of thesecond C_(H)3 domain to protein A. Various such modifications aredescribed in, e.g., U.S. Application Publication Nos. 2010/0331527 and2011/0195454, incorporated herein by reference.

One method for making an epitope-binding protein that binds more thanone epitope and exhibits pH-dependent epitope binding property is toimmunize a first mouse in accordance with the invention with an antigenthat comprises a first epitope of interest, wherein the mouse comprises(1) an endogenous immunoglobulin light chain variable region locus thatdoes not contain an endogenous mouse light chain variable region genesequence that is capable of rearranging and forming a light chain,wherein at the endogenous mouse immunoglobulin light chain variableregion locus is a single rearranged human light chain variable regionoperably linked to the mouse endogenous light chain constant regiongene, and, in some embodiments, the rearranged human light chainvariable region is selected from a human Vκ1-39Jκ5 and a human Vκ3-20Jκ1comprising a substitution of at least one non-histidine codon with ahistidine codon, and (2) the endogenous mouse V_(H) gene segments havebeen replaced in whole or in part with human V_(H) gene segments, suchthat immunoglobulin heavy chains made by the mouse are solely orsubstantially heavy chains that comprise human variable domains andmouse constant domains. When immunized, such a mouse will make a reversechimeric antibody, comprising only one of two human light chain variabledomains (e.g., one of human Vκ1-39Jκ5 or human Vκ3-20Jκ1, e.g.,comprising a substitution of at least one amino acid with a histidine).Commonly, at least some of the substituted histidine residues introducedinto the germline sequence will be retained in the reverse chimericantibody. Once a B cell is identified that encodes a heavy chainvariable domain that binds the epitope of interest and expresses anantibody that exhibits pH-dependent antigen binding properties, thenucleotide sequence of the heavy chain variable region (and, optionally,the light chain variable region) can be retrieved (e.g., by PCR) andcloned into an expression construct in frame with a suitable humanimmunoglobulin heavy chain constant region sequence. This process can berepeated to identify a second heavy chain variable domain that binds asecond epitope, and a second heavy chain variable region gene sequencecan be retrieved and cloned into an expression vector in frame to asecond suitable human immunoglobulin heavy chain constant regionsequence. The first and the second immunoglobulin constant domainsencoded by the constant region gene sequence can be the same ordifferent isotype, and one of the immunoglobulin constant domains (butnot the other) can be modified as described herein or in US2010/0331527A1, and epitope-binding protein can be expressed in asuitable cell and isolated based on its differential affinity forProtein A as compared to a homodimeric epitope-binding protein, e.g., asdescribed in US 2010/0331527A1.

Thus, in various embodiments, following isolation of the DNA andselection of the first and second nucleic acid sequences that encode thefirst and second human heavy chain variable domains having the desiredspecificities/affinities, and a third nucleic acid sequence that encodesa human light chain domain (a germline rearranged sequence or a lightchain sequence isolated from a non-human animal as described herein) andcomprises a substitution of at least one non-histidine codon with ahistidine codon, the three nucleic acids sequences encoding themolecules are expressed to form the bispecific antibody usingrecombinant techniques which are widely available in the art. Often, theexpression system of choice will involve a mammalian cell expressionvector and host so that the bispecific antibody is appropriatelyglycosylated (e.g., in the case of bispecific antibodies comprisingantibody domains which are glycosylated). However, the molecules canalso be produced in the prokaryotic expression systems. Normally, thehost cell will be transformed with DNA encoding both the first humanheavy chain variable domain, the second human heavy chain variabledomain, the human light chain domain on a single vector or independentvectors. However, it is possible to express the first human heavy chainvariable domain, second human heavy chain variable domain, and humanlight chain domain (the bispecific antibody components) in independentexpression systems and couple the expressed polypeptides in vitro. Invarious embodiments, the human light chain domain is derived from agermline sequence but for the substitution of at least one non-histidinecodon with a histidine codon, e.g., in a CDR codon. In variousembodiments, the human light chain domain comprises no more than one, nomore than two, no more than three, no more than four, or no more thanfive somatic hypermutations within the light chain variable sequence ofthe light chain domain. In some embodiments, the somatic hypermutationsdo not alter the presence of at least one histidine residue introducedinto the germline sequence of the light chain variable region.

In various embodiments, the nucleic acid(s) (e.g., cDNA or genomic DNA)encoding the two heavy chains and single human light chain with asubstitution of at least one non-histidine with a histidine is insertedinto a replicable vector for further cloning (amplification of the DNA)and/or for expression. Many vectors are available, and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Each component may be selected individually or based on a host cellchoice or other criteria determined experimentally. Several examples ofeach component are known in the art.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid sequences that encode each or all the components of the bispecificantibody. A large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto bispecific antibody-encoding DNA by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the bispecific antibody components.Suitable expression vectors for various embodiments include those thatprovide for the transient expression in mammalian cells of DNA encodingthe bispecific antibody. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of bispecific antibodieshaving desired binding specificities/affinities or the desired gelmigration characteristics relative to the parental antibodies havinghomodimers of the first or second human heavy chain variable domains.

In various embodiments, once the DNA encoding the components of thebispecific antibody are assembled into the desired vector(s) asdescribed above, they are introduced into a suitable host cell forexpression and recovery. Transfecting host cells can be accomplishedusing standard techniques known in the art appropriate to the host cellselected (e.g., electroporation, nuclear microinjection, bacterialprotoplast fusion with intact cells, or polycations, e.g., polybrene,polyornithine, etc.).

A host cell is chosen, in various embodiments, that best suits theexpression vector containing the components and allows for the mostefficient and favorable production of the bispecific antibody species.Exemplary host cells for expression include those of prokaryotes andeukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In various embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In various embodiments,the cell is eukaryotic cell selected from CHO (e.g., CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI138, MRC 5,Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertolicell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cellline derived from an aforementioned cell. In various embodiments, thecell comprises one or more viral genes, e.g. a retinal cell thatexpresses a viral gene (e.g., a PER.C6™ cell).

Mammalian host cells used to produce the bispecific antibody may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. Media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other supplements may also be includedat appropriate concentrations as known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are, invarious embodiments, those previously used with the host cell selectedfor expression, and will be apparent to those skilled in the art.

The bispecific antibody may be recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysate when directly produced without a secretory signal. If thebispecific antibody is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g., Triton-X 100).

Following isolation, a bispecific antibody comprising a two human heavychains and a single human light chain derived from a rearranged humanlight chain variable region gene sequence, the sequence selected fromVκ1-39Jκ5 and Vκ3-20Jκ1 sequences that comprise a substitution of atleast one non-histidine codon with a histidine codon, is screened forits ability to exhibit pH dependent binding to one, preferably both ofits antigens. The ability of bispecific antibodies to bind its antigensdifferently at neutral and acidic pH's (e.g., their ability todemonstrate decreased t_(1/2) at acidic pH compared to neutral pH) canbe determined by a variety of techniques available in the art anddescribed in the following examples, e.g., BIACORE™ assay.

A similar method for making a binding protein that binds more than oneepitope and exhibits pH-dependent epitope binding property, by utilizinga light chain derived from a mouse comprising no more than two humanV_(L) and one or more, e.g., two or more, human J_(L) gene segments withhistidine modifications, and heavy chain(s) derived from the same ordifferent mouse (e.g., a universal light chain mouse) is also provided,and would be apparent from the present disclosure. Briefly, micedescribed herein (e.g., mice comprising a dual light chain locus) may behumanized with an antigen of interest and a light chain and/or heavychain variable domain from a B cell that binds the epitope of interestmay be identified, and the nucleotide sequence cloned in frame into avector comprising a suitable constant region; same process is repeatedto obtain other variable domains of interest, and variable domainsco-expressed in a suitable cell line as described in more detail above.

Additional Methods for Generating Antigen-Binding Proteins withpH-Dependent Antigen Binding

Various methods of generating antigen-binding proteins with pH-dependentantigen binding properties in genetically modified non-human animalsdescribed herein are provided. Also provided are methods of generatingantigen binding proteins with pH-dependent antigen binding properties invitro. Such methods may involve generating various components of theantigen-binding proteins in vivo in genetically modified non-humananimals, and then modifying them and reassembling them in vitro outsidean organism as protein complexes expressed in mammalian cell culture.

In one embodiment, the method of generating antigen-binding proteinswith pH-dependent antigen binding properties utilizes an antigen-bindingprotein sequence, e.g., an antibody sequence, that is generated in amouse comprising a limited repertoire of light chain variable region Vand J segments, e.g., human light chain variable region V and Jsegments, “universal light chain” or “common light chain” mouse (“ULC”mouse), such as the mouse described in U.S. Application Publication Nos.2011/0195454, 2012/0021409, 2012/0192300 2013/0045492, and 2013/0185821,all incorporated herein by reference. In one embodiment, the method ofgenerating antigen-binding proteins with pH-dependent antigen bindingproperties utilizes an antigen binding protein sequence that isgenerated in a mouse comprising a single rearranged human light chainvariable region gene sequence. In one embodiment, the method utilizes anantigen binding protein generated in a mouse comprising a singlerearranged human light chain variable region gene sequence selected fromhuman Vκ1-39Jκ5 and human Vκ3-20Jκ1. In another embodiment, the methodof generating antigen-binding proteins with pH-dependent antigen bindingproperties utilizes an antigen-binding protein sequence generated in alimited variable gene segment mouse, e.g., a dual light chain mouse.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting a first antibody that binds to an antigen ofinterest (e.g., binds to an antigen of interest with a desiredaffinity), modifying an immunoglobulin light chain nucleotide sequenceof the first antibody to comprise a substitution of at least onenon-histidine codon with a histidine codon, expressing an immunoglobulinheavy chain of the first antibody and the modified immunoglobulin lightchain in a cell, and selecting a second antibody expressed in the cellthat retains binding to the antigen of interest (e.g., retains desiredaffinity for the antigen of interest) at neutral pH and displays reducedbinding to the antigen of interest at an acidic pH.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting an immunoglobulin heavy chain from an antibody(e.g., obtained from a non-human animal, e.g., a mouse, e.g., a ULCmouse) that comprises an immunoglobulin light chain having a singlerearranged human immunoglobulin light chain variable region sequencewherein the antibody binds to an antigen of interest (e.g., binds to anantigen of interest with a desired affinity); modifying the nucleic acidsequence of the immunoglobulin light chain such that the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon; expressing the selected immunoglobulin heavy chain andthe immunoglobulin light chain comprising the substitution of at leastone amino acid with a histidine in its variable domain; and selecting anantibody that retains binding to the antigen of interest at a neutral pH(e.g., retains desired affinity to the antigen of interest) whiledisplaying reduced binding to the antigen of interest at an acidic pH.In various embodiments, the immunoglobulin heavy chain is derived from arearrangement of human heavy chain variable gene segments (human V, D,and J segments).

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH-dependent antigen binding propertiescomprises (1) immunizing a non-human animal, e.g., a mouse, comprising asingle rearranged human light chain variable region gene sequence and arepertoire of unrearranged human heavy chain variable gene segments (V,D, and J segments) with an antigen of interest and allowing a mouse tomount an immune response to said antigen, (2) selecting in the non-humananimal, e.g., in the mouse, an antibody that binds to the antigen ofinterest with a desired affinity, (3) isolating from the non-humananimal, e.g., from the mouse, a nucleotide sequence of an immunoglobulinheavy chain of the antibody that binds to the antigen of interest with adesired affinity, (4) determining the nucleotide sequence of said heavychain, (5) modifying a nucleotide sequence of an immunoglobulin lightchain containing the single rearranged human immunoglobulin light chainvariable region to comprise a substitution of at least one non-histidinecodon with a histidine codon, (6) expressing the immunoglobulin heavychain of the antibody that binds to the antigen of interest with desiredaffinity and the immunoglobulin light chain comprising the histidinemodification in a cell, and (7) determining whether the antibodyexpressed in the cell retains binding to the antigen at a neutral pHwhile displaying reduced binding at an acidic pH. In one embodiment, theantibody expressed in the cell exhibits desired affinity to the antigenat neutral pH. In various embodiments, the immunoglobulin heavy chain isderived from a rearrangement of human heavy chain variable gene segments(human V, D, and J segments). In another embodiment, also providedherein is a similar method for generating an antigen-binding proteinwith pH-dependent binding properties wherein instead of immunizing amouse comprising a single rearranged human light chain variable regionsequence, the method comprises immunizing a mouse comprising a limitedrepertoire of light chain variable gene segments, e.g., a mousecomprising no more than two human V_(L) gene segments and a plurality,e.g., two or more, human J_(L) gene segments.

In one embodiment, the mouse comprising a single rearranged human lightchain variable region gene sequence is a universal light chain or commonlight chain “ULC” mouse described in, e.g., U.S. Application PublicationNos. 2011/0195454, 2012/0021409, 2012/0192300, 2013/0045492, and2013/0185821. In one embodiment, the single rearranged human light chainvariable region gene sequence is selected from human Vκ1-39Jκ5 and humanVκ3-20Jκ1 sequence.

In one embodiment, the antigen of interest is selected from a solubleantigen, a cell surface antigen (e.g., a tumor antigen) and a cellsurface receptor. In a specific embodiment, the cell surface receptor isan immunoglobulin receptor. In a specific embodiment, the immunoglobulinreceptor is an Fc receptor.

In one embodiment, the desired affinity of an antibody for an antigenexpressed as a dissociation constant (K_(D)) at a neutral pH is lessthan 10⁻⁶ M, e.g., less than 10⁻⁸M, e.g., less than 10⁻⁹ M, e.g., lessthan 10⁻¹⁰ M, e.g., less than 10⁻¹¹ M, e.g., less than 10⁻¹² M.

As explained above, the ULC mice, in one embodiment, comprise a singlerearranged human immunoglobulin light chain variable gene sequence, andexpress antibodies in response to the antigen where the affinity ofantibodies to the antigen is primarily mediated through the heavy chainsof their antibodies. These mice comprise a repertoire of human heavychain variable (V, D, and J) segments, that rearrange to encode a humanheavy chain variable domain of an antibody that also comprises the lightchain derived from the single rearranged human light chain variablesequence. In one embodiment, upon antigen exposure, these mice utilizethe diverse repertoire of human heavy chain variable (V, D, and J)segments to generate an antibody with affinity to and specificity forthe antigen. Thus, upon exposure to the antigen, the nucleotide sequenceof an immunoglobulin heavy chain of the antibody generated in the ULCmice may be isolated and utilized to generate a desired binding proteinalso comprising an immunoglobulin light chain derived from the singlerearranged human immunoglobulin light chain variable region sequence(e.g., the single rearranged human immunoglobulin light chain variableregion sequence with a substitution of at least one non-histidine codonwith a histidine codon).

In one embodiment of the ULC mice, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments. Thus, the ULC mouse utilizes a diverse repertoire of humanvariable region gene segments (V, D, and J segments) to generate anantibody in response to the antigen of interest.

Once the heavy chain of the antibody that binds to the antigen ofinterest with the desired affinity is determined, the nucleotidesequence of the heavy chain is isolated and sequenced. The sequence iscloned into a vector for expression in suitable host cells, e.g.,eukaryotic cells, e.g., CHO cells. In one embodiment, the sequence of ahuman heavy chain constant region is cloned downstream of the humanheavy chain variable region sequence isolated from the mouse (e.g., fromthe ULC mouse).

In one embodiment, the method of generating an antigen-binding proteinwith pH-dependent antigen-binding properties comprises modifying anucleotide sequence of the immunoglobulin light chain, particularly thesequence of the single rearranged human immunoglobulin light chainvariable region, to comprise a substitution of at least onenon-histidine codon with a histidine codon. Various techniques formodifying a nucleotide sequence are known in the art, e.g., sitedirected mutagenesis. In addition, a nucleotide sequence comprising thedesired histidine substitution may be synthesized de novo.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon comprises a substitution resulting in expressionof one, two, three, four, or more histidine residues. In one embodiment,the substitution(s) results in expression of three or four histidineresidues. In one embodiment, the substitution(s) is in theimmunoglobulin light chain variable region. In one embodiment, thesubstitution(s) is in the CDR codon, e.g., CDR1, CDR3, and/or CDR3codon. In one embodiment, the substitution(s) is in the CDR3 codon.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ1-39Jκ5 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of a histidineat position selected from 105, 106, 108, 111, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 108, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 108 and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 108. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 108,and 111. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 108, and 111. In one embodiment, amino acidand nucleic acid sequences of Vκ1-39Jκ5 CDR3 regions comprising varioushistidine substitutions are depicted in FIG. 2 and included in thesequence listing.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ3-20Jκ1 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of a histidineat position selected from 105, 106, 107, 109, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 107, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 107 and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 107. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 107,and 109. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 107, and 109. Selected amino acid andnucleic acid sequences of Vκ3-20Jκ1 CDR3 regions comprising varioushistidine substitutions are depicted in FIG. 12 and included in thesequence listing.

Once the sequence of immunoglobulin light chain, e.g., humanimmunoglobulin light chain variable domain, is modified to includehistidine residues at desired positions, the nucleotide sequence of thelight chain is cloned into a vector for expression in suitable hostcells, e.g., eukaryotic cells, e.g., CHO cells. In one embodiment, thesequence of a human light chain constant region is cloned downstream ofthe modified nucleotide sequence of human variable region.

In one embodiment, vectors comprising nucleotide sequence encodingmodified human immunoglobulin light chain and selected humanimmunoglobulin heavy chain are co-expressed in a suitable host cell,e.g., eukaryotic host cell, e.g., CHO cell, to generate anantigen-binding protein. Various host cells that can be used forexpression are known in the art and are mentioned throughout thisspecification.

An antigen-binding protein, e.g., an antibody, generated in the hostcell may be secreted into cell supernatant, which is screened for properexpression and affinity for the original antigen at neutral pH. Theantigen-binding protein may also be recovered from cell lysate, or, ifmembrane bound, released from the membrane using a suitable detergent(e.g., Triton-X). The antigen-binding protein with desiredcharacteristics may be purified.

In one embodiment, the antigen-binding protein comprising histidinemodification(s) retains the affinity to the antigen that is comparableto the affinity to the antigen of the same (original) antigen-bindingprotein that does not comprise histidine modification(s). In oneembodiment, the affinity of the histidine-modified antigen-bindingprotein for the antigen of interest expressed as a dissociation constant(K_(D)) at a neutral pH is less than 10⁻⁶ M, e.g., less than 10⁻⁸M,e.g., less than 10⁻⁹ M, e.g., less than 10⁻¹⁰ M, e.g., less than 10⁻¹¹M, e.g., less than 10⁻¹² M.

In one embodiment, the antigen-binding protein, e.g., an antibody,comprising histidine modifications described herein exhibits pHdependent antigen binding properties. In one embodiment, theantigen-binding protein comprising histidine modifications possessesenhanced pH dependent properties over an equivalent antigen-bindingprotein without the histidine modifications (antigen-binding protein ofthe same amino acid sequence but for the histidine modifications). Inone embodiment, the antigen-binding protein described herein retainsbinding to the antigen at neutral pH (e.g., retains desired affinity forthe antigen at neutral pH) while displaying reduced binding at an acidicpH. In one embodiment, the antigen-binding protein, e.g., the antibody,described herein, exhibits no binding to the antigen in acidic pH, whileretaining binding to the antigen at neutral pH. In one embodiment, anantigen-binding protein described herein, has a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to the dissociativehalf-life (t_(1/2)) of the antigen-binding protein at a neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, an antigen-binding protein described herein has at_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein described herein has a t_(1/2) atan acidic pH and 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein described herein has a t_(1/2) at an acidic pHand 25° C. of about 2 min or less. In one embodiment, an antigen-bindingprotein described herein has a t_(1/2) at an acidic pH and 25° C. ofless than about 1 min.

In one embodiment, the antigen-binding protein e.g., the antibody,comprising histidine modifications described herein, exhibits increasedserum half life upon administration of a therapeutic dose to a subjectas compared to a serum half life upon administration of an equivalenttherapeutic dose of antigen-binding protein that does not comprisehistidine modifications (e.g., the original antigen-binding protein thatdoes not comprise histidine modifications). In some embodiments, theincrease in serum half life upon administration of a dose of theantigen-binding protein comprising histidine modifications describedherein over a serum half life upon administration of the same dose ofthe antigen-binding protein not comprising histidine modifications isabout 2 fold, e.g., about 5 fold, e.g., about 10 fold, e.g., about 15fold, e.g., about 20 fold, or greater. In one embodiment, serumhalf-life is at least about 1 day, e.g., at least about 2 days, e.g., atleast about 7 days, e.g., at least about 14 days, e.g., at least about30 days, e.g., at least about 60 days.

In addition to the in vitro methods for generating antigen-bindingproteins with pH-dependent antigen binding properties described above,also provided herein are antigen-binding proteins, e.g., antibodies,generated by said method. In addition, said method may be utilized togenerate multi-specific, e.g., bispecific, antigen-binding proteins, byselecting two different human immunoglobulin heavy chains that bind to acommon (universal) light chain in a mouse, determining nucleotidesequences of the heavy chains, modifying universal light chain tocomprise histidine substitutions as described above, and co-expressingtwo human heavy chains with a single histidine-modified universal lightchain in a host cell. Various steps for generating an antigen-bindingprotein described above may be applicable to the method of generating abispecific antigen-binding protein. Bispecific antigen binding protein,confirmed to possess desired affinity for the antigen(s) andpH-dependent antigen binding properties may be purified. Thus,bispecific antibodies comprising two human heavy chains and a singlehuman light chain comprising a human light chain variable domainsequence encoded by a human variable region gene, e.g., Vκ1-39Jκ5 orVκ3-20Jκ1 variable region gene comprising a substitution of at least onenon-histidine codon with a histidine codon, is provided.

Also in some embodiments provided herein are methods for generatingantigen-binding proteins with pH-dependent binding properties utilizinglight chains, e.g., antigen-specific light chains, generated in duallight chain mice; as well as antigen-binding proteins, e.g., antibodies,generated by said methods. Such methods and antibodies generated by saidmethods would be apparent from the present specification.

Also in some embodiments provided are constructs utilized in making anantigen-binding protein comprising human immunoglobulin heavy chain andhuman immunoglobulin light chain comprising histidine substitutions.Host cells expressing antigen-binding proteins, e.g., antibodies,described herein are also provided.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for. TheExamples do not include detailed descriptions of conventional methodsthat would be well known to those of ordinary skill in the art(molecular cloning techniques, etc.). Unless indicated otherwise, partsare parts by weight, molecular weight is average molecular weight,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1 Identification of Histidine Residues in Antigen-Specific HumanLight Chains

Generation of a common light chain mouse (e.g., Vκ1-39 or Vκ3-20 commonlight chain mouse) and antigen-specific antibodies in those mice isdescribed in, e.g., U.S. patent application Ser. Nos. 13/022,759,13/093,156, and 13/412,936 (Publication Nos. 2011/0195454, 2012/0021409,and 2012/0192300, respectively), incorporated by reference herein intheir entireties. Briefly, rearranged human germline light chaintargeting vector was made using VELOCIGENE® technology (see, e.g., U.S.Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech. 21(6): 652-659) to modify mouse genomicBacterial Artificial Chromosome (BAC) clones, and genomic constructswere engineered to contain a single rearranged human germline lightchain region and inserted into an endogenous κ light chain locus thatwas previously modified to delete the endogenous κ variable and joininggene segments. Targeted BAC DNA was then used to electroporate mouse EScells to create modified ES cells for generating chimeric mice thatexpress a rearranged human germline Vκ1-39Jκ5 or Vκ3-20Jκ1 region.Targeted ES cells were used as donor ES cells and introduced into an8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S.Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice thatare essentially fully derived from the donor gene-targeted ES cellsallowing immediate phenotypic analyses Nature Biotech. 25(1): 91-99).VELOCIMICE® independently bearing an engineered human germline Vκ1-39Jκ5or Vκ3-20Jκ1 light chain region were identified by genotyping using amodification of allele assay (Valenzuela et al., supra) that detects thepresence of the unique rearranged human germline light chain region.

Mice bearing an engineered human germline light chain locus (ULC mice)were bred with mice that contain a replacement of the endogenous mouseheavy chain variable gene locus with the human heavy chain variable genelocus (see U.S. Pat. No. 6,596,541; the VELOCIMMUNE® mouse, RegeneronPharmaceuticals, Inc.). VELOCIMMUNE® mouse containing a singlerearranged human germline light chain region is challenged with anantigen of interest and antibodies comprising a universal light chain(e.g., Vκ1-39Jκ5) are isolated and sequenced.

Amino acid sequences of selected light chains (A-K, corresponding to SEQID NOs:136-146, respectively) containing Vκ1-39 from antigen-specifichuman antibodies were aligned. Histidine mutations in the CDRs of humanVκ1-39-derived light chains for a selected number of antigen-specifichuman antibodies were identified (FIG. 1). The amino acid sequence ofgermline Vκ1-39 is shown above the alignments and set forth in SEQ IDNO:1, the complete variable domain amino acid sequence for Vκ1-39Jκ5 isset forth in SEQ ID NO:80.

Example 2 Engineering and Characterization of Histidine-SubstitutedHuman Universal Light Chain Antibodies Example 2.1 Engineering ofHistidine Residues into a Germline Human Rearranged Light Chain

Histidine residues were engineered into a rearranged human Vκ1-39Jκ5light chain using site directed mutagenesis primers specificallydesigned to introduce engineered histidine residues at Q105, Q106, Y108,and P111 positions of the human Vκ1-39Jκ5 light chain. Site directedmutagenesis was performed using molecular techniques known in the art(e.g., QuikChange II XL Site Directed Mutagenesis Kit, AgilentTechnologies). Locations of the engineered residues in the CDR3 areshown in FIG. 2, the nucleic acid sequences of histidine-substitutedCDR3's depicted in FIG. 2 are set forth in SEQ ID NOs: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 (corresponding amino acidsequences are set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, and 33). The nucleic acid and amino acid sequencesof germline rearranged Vκ1-39Jκ5 CDR3 are set forth in SEQ ID NOs: 2 and3, respectively.

Example 2.2 Construction and Expression of Histidine Engineered LightChains

Human Vκ1-39-derived light chains containing germline engineeredhistidine residues made according to Example 2 were constructed andpaired with various human heavy chains (labeled 1-5), specific for ahuman cell surface receptor, to analyze expression in CHO cells. Thefive human heavy chains specific for a human cell surface receptor thatwere paired with histidine-substituted Vκ1-39-derived light chains wereobtained from mice that have a single rearranged human light chain (ahuman Vκ1-39/Jκ5 rearranged light chain; see US2011/0195454A1).

Enzyme-Linked Immunosorbent Assay (ELISA):

Antibody secretion from CHO cells was detected using an Fc ELISA, forlight chains with indicated histidine modifications with five differentheavy chains. The light and heavy chain sequences (but for themodifications) were generated in mice that have a single rearrangedhuman light chain (e.g., a human Vκ1-39/Jκ5 rearranged light chain; seeUS2011/0195454A1). Capture antibody was goat anti-human IgG anddetection antibody was goat anti-human (Fc gamma-specific)-HRP. Theresults are shown in FIG. 3. ULC+heavy: specific heavy chain andunmodified human Vκ1-39-derived light chain. As shown in FIG. 3,expression was detected in about all mutants.

Protein Immunoblot.

Expression in supernatants of CHO cells of paired antigen-specific heavychains with histidine engineered light chains was further analyzed bywestern blot. Samples were run on a 4-12% tris-glycine gel. Resultsusing a selected heavy chain (heavy chain 3) are shown in FIG. 4. ULCrefers to a rearranged human Vκ1-39-derived light chain (as describedabove).

Example 2.3 Determination of Binding Affinity of Histidine EngineeredLight Chains

Equilibrium dissociation constants (K_(D)), dissociative half-lives(t_(1/2)), and other kinetic parameters for selected antibodysupernatants were determined by SPR (Surface Plasmon Resonance) using aBIACORE™ T200 instrument (GE Healthcare). Kinetics were measured at pH7.4 and at pH 5.75. Results are shown in FIGS. 5A-5E.

Numerical values for the kinetic binding properties (e.g., k_(a), k_(d),K_(D), t_(1/2), etc.) of antibodies binding to immunogen at neutral pH(pH 7.4) and at acidic pH (pH 5.75) were obtained using a real-timesurface plasmon resonance biosensor (Biacore T200.) A Biacore CM5 sensorchip was derivatized with a mouse anti-human Fc antibody to captureantibodies from the supernatant. A single concentration (50 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 2.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 8 minutes. Kinetic association (ka) and dissociation(kd) rate constants were determined by processing and fitting the datato a 1:1 binding with a mass transport model using Biacore T200Evaluation software version 1.0. Equilibrium dissociation constants(K_(D)) and dissociative half-lives (t_(1/2)) were calculated from thekinetic rate constants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=(ln2/(60*k_(d)).

As shown in FIG. 5, in a binding assay of antibody to a cell surfacereceptor, two out of five antibodies with histidine-modified commonlight chains (histidine modified CDR3's of Vκ1-39/Jκ5 light chains) thatwere paired with the antigen-specific human heavy chains, exhibitedbinding to the antigen (e.g., to a cell surface receptor) with differentaffinities at pH 7.4 and pH 5.75. Antibodies with histidinemodifications that retain binding at pH 7.4, but that exhibit a lowbinding or no detectable binding at pH 5.75, are desirable. Antibodieswith histidine modification that exhibit reduced t_(1/2) at pH 5.75 ascompared to pH 7.4 are desirable.

Antigen binding data for three antibodies comprising histidine-modifiedcommon light chains and three antigen-specific heavy chains (labeled 2,3, and 6) at different pHs is summarized further in FIG. 6. Theseantibodies exhibited significant drop in antigen binding at pH 5.75 incomparison to pH 7.4, as demonstrated, e.g., by reduction in t_(1/2) orno binding detected at pH 5.75.

Example 3 Engineering and Characterization of Genetically Modified MouseComprising a Human Histidine-Substituted Vκ1-39Jκ5 Universal Light ChainExample 3.1 Constructing of Targeting Vector for Engineering HistidineResidues in a Rearranged Human Light Chain Variable Region

A genetically modified mouse containing a rearranged human light chaingene having histidine residues engineered into a CDR region of the humanlight chain is made using targeting vectors made by standard molecularcloning techniques known in the art.

Briefly, various rearranged human germline light chain targeting vectorsare made using VELOCIGENE® technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al. (2003) High-throughput engineering ofthe mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659) to modify mouse genomic BacterialArtificial Chromosome (BAC) DNA to contain a single rearranged humangermline light chain region and inserted into an endogenous κ lightchain locus that was previously modified to delete the endogenous κvariable and joining gene segments. The rearranged human germline lightchain region is modified at one or more nucleotide positions within thesequence of the light chain to encode histidine residues that are notnormally present at the respective locations of the germline sequence.The targeting vectors are electroporated into mouse embryonic stem (ES)cells and confirmed using a quantitative PCR assay (e.g., TAQMAN™).

Specifically, a strategy for constructing these targeting vectors isshown in FIGS. 8A-8F. A plasmid used for generating a targeting vectorfor common (universal) light chain mouse (“ULC mouse,” described in,e.g., US2011/0195454A1), containing pBS+FRT-Ub-Hyg-FRT+ mouse Vκ3-7leader+human Vκ1-39Jκ5 was modified by site directed mutagenesis(QuickChange II XL Kit) to replace Q105, Q106, Y108 and P111 or Q106,Y108 and P111 with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 7 (See FIG. 8A for thisengineering step). Resultant vectors (H105/106/108/111 and H106/108/111)were modified further and ligated into a vector comprising mouse Igκconstant region, mouse enhancers, a mouse 3′ homology arm and a SPECcassette (FIG. 8B). Further modification involved ligation into a vectorcarrying 5′ mouse arm and comprising Frt-Ub-NEO-Frt cassette (FIG. 8B).Resultant targeting vectors were electroporated into ES cells comprisingdeletion of the mouse Igκ variable locus (comprising κ variable andjoining gene segments) (FIGS. 8C-8F).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ1-39Jκ5 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 1 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 8C-8F.

TABLE 2 Primers and Probes Used for ES Cell Screening Probe Name AssayProbe Sequence 5′ Primer 3′ Primer Neo GOA TGGGCACAACAGACGGTGGAGAGGCTATT GAACACGGCGGCAT AATCGGCTG CGGC CAG (SEQ ID NO: 38)(SEQ ID NO: 39) (SEQ ID NO: 40) ULC-m1 GOA CCATTATGATGCTCAGGTGAGGGTACAGA TGACAAATGCCCTAA CATGCCTCTCTGTT TAAGTGTTATGAGTTATAGTGATCA C (SEQ ID NO: 42) (SEQ ID NO: 43) (SEQ ID NO: 41) 1633h2GOA ATCAGCAGAAACCAG GGGCAAGTCAGAGCA TGCAAACTGGATGCA (Vκ1-39Jκ5-GGAAAGCCCCT TTAGCA GCATAG specific) (SEQ ID NO: 44) (SEQ ID NO: 45)(SEQ ID NO: 46) mlgKd2 Retention GGCCACATTCCATGG GCAAACAAAAACCACCTGTTCCTCTAAAAC GTTC TGGCC TGGACTCCACAGTAA (SEQ ID NO: 47)(SEQ ID NO: 48) ATGGAAA (SEQ ID NO: 49) mlgKp15 RetentionGGGCACTGGATACGA CACAGCTTGTGCAGC AGAAGAAGCCTGTAC TGTATGG CTCCTACAGCACTAAGTTT (SEQ ID NO: 50) (SEQ ID NO: 51) TACAGTCA (SEQ ID NO: 52)

The NEO selection cassette introduced by the targeting constructs wasdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 8C and 8E). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence were made from the targeted EScells described above.

Pups were genotyped and pups heterozygous for the engineeredhistidine-modified human light chain were selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H106/108/111;“1930”) or four (H105/105/108/111; “1927”) histidine modifications arelisted in Table 2 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 2 Primers and Probes Used for Genotyping Probe Name AssayProbe Sequence 5′ Primer 3′ Primer 1927jxn3 GOA 1927 (4 ACCATAGTCACAGTAAGCAGTCTGCAACCT CCCTTGGCCGAAGGT His) mouse- CCCA GAAGATTT GAT specific(SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55) 1930jxn3 GOA 1930 (3ATAGTCACAGTACCC AGTCTGCAACCTGAA CCCTTGGCCGAAGGT His) mouse- ATCCGATTTTGC GAT specific (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58)

Example 3.2 Analysis of Immune Response to Antigen in Mice with HumanHistidine-Substituted Universal Light Chains

Cell surface receptor (“Antigen A”) was used as the immunogen toimmunize mice that were either heterozygous for expression of apre-arranged human kappa light chain utilizing Vκ1-39 and Jκ5 that has 4histidine substitutions in CDR3 (hereinafter “HULC 1927”) orheterozygous for expression of a pre-arranged human kappa light chainutilizing Vκ1-39 and Jκ5 that has 3 histidine substitutions in CDR3(hereinafter “HULC1930”), or homozygous WT mice. Pre-immune serum wascollected from the mice prior to the initiation of immunization. Theimmunogen was administered at 2.35 μg of protein for the initial primingimmunization mixed with 10 μg of CpG oligonucleotide as an adjuvant(Invivogen) in a volume of 25 μl via footpad (f.p.). Subsequently, micewere boosted via the same route with 2.35 μg of Antigen A along with 10μg of CpG and 25 μg of Adju-Phos (Brenntag) as adjuvants on days 3, 6,11, 13, 17, 20 for a total of 6 boosts. The mice were bled on days 15and 22 after the 4^(th) and 6^(th) boost, respectively. Their antiserumwas assayed for antibody titers to Antigen A.

Antibody serum titers against immunogen were determined by a standardELISA. To perform the ELISA, 96-well microtiter plates (ThermoScientific) were coated at 2 μg/ml with Antigen A in phosphate-bufferedsaline (PBS, Irvine Scientific) overnight at 4° C. The next day, plateswere washed with phosphate-buffered saline containing 0.05% Tween 20(PBS-T, Sigma-Aldrich) four times using a plate washer (MolecularDevices). Plates were then blocked with 250 μl of 0.5% bovine serumalbumin (BSA, Sigma-Aldrich) in PBS and incubated for 1 hour at roomtemperature. The plates were then washed four times with PBS-T. Serafrom immunized mice and pre-immune sera were serially diluted three-foldin 0.5% BSA-PBS starting at 1:300 or 1:1000, added to the blocked platesin duplicate, and then incubated for 1 hour at room temperature. Thelast two wells were left blank to be used as a secondary antibodycontrol (background control). The plates were again washed four timeswith PBS-T in a plate washer. Goat anti-mouse IgG-Fc-Horse RadishPeroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch)was then added to the plates at 1:5000/1:10,000 dilution and incubatedfor 1 hour at room temperature. Plates were then washed eight times withPBS-T and developed using TMB/H₂O₂ as substrate. The substrate wasincubated for 20 min and the reaction was stopped with 2 N sulfuric acid(H₂SO₄, VWR, cat# BDH3500-1) or 1 N phosphoric acid (JT Baker,Cat#7664-38-2). Plates were read on a spectrophotometer (Victor, PerkinElmer) at 450 nm. Antibody titers were computed using Graphpad PRISMsoftware.

The immune response induced in mice to the injected immunogen isrepresented as antibody titers, which is defined as the reciprocal ofthe highest serum dilution at which antigen binding absorbance istwo-fold higher over background. Therefore, the higher the number, thegreater the humoral immune response to the immunogen. Antibody titersinduced to the immunogen were very high in both strains of HULC mice andin the WT mice, with no significant differences observed among thestrains (FIG. 9).

Example 3.3 Generation of pH-Sensitive Monoclonal Antibodies

When a desired immune response to the immunogen was achieved in bothstrains of HULC mice and in the WT mice, splenocytes from each mousestrain were harvested and fused with mouse myeloma cells to generatehybridoma cells, which were allowed to grow in 96-well plates. After 10days of growth, supernatants from each hybridoma cell-containing wellwere screened via immunogen-specific ELISA to identify positive antigenbinding samples. For the ELISA, 96 well micro-titer plates were coatedwith 1 μg/mL of an anti-myc polyclonal antibody (Novus Biologicals,#NB600-34) overnight at 4° C. to immobilize the myc-tagged antigen,followed by blocking with a solution of 0.5% (w/v) BSA in PBS. Theplates were washed, the antigen solutions were added to the plates at aconcentration of 1 μg/mL and allowed to bind to the coated plate for 1hour at room temperature. Subsequently, supernatants from hybridomacells were added to the wells at 1:50 dilution and allowed to bind for 1hour at room temperature. The plate bound antibodies were detected usingan anti-mouse IgG polyclonal antibody conjugated with HRP (JacksonImmunoresearch, #115-035-164). TMB substrates were added to the plates(BD Biosciences, #51-2606KC/51-2607KC) and colorimetric signals weredeveloped according to manufacturer recommended protocol. The absorbancewas recorded at 450 nm on a Victor Wallac plate reader. Antigen positivesamples defined as having an OD equal to or greater than 0.5 (with thebaseline having OD of about 0.1) were subject to affinity screeningusing a real-time surface plasmon resonance biosensor (Biacore 4000).

Kinetic binding parameters (e.g., k_(a), k_(d), K_(D), t_(1/2), etc.)for antibody binding to the immunogen at neutral pH (pH 7.4) and atacidic pH (pH 6.0) were recorded. A Biacore CM4 sensor chip wasderivatized with a polyclonal goat anti-mouse Fc antibody to captureantibodies from the supernatant. A single concentration (100 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 1.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 2.5 minutes. Kinetic association (k_(a)) anddissociation (k_(d)) rate constants were determined by processing andfitting the data to a 1:1 binding with a mass transport model usingBiacore 4000 Evaluation software version 1.0. Equilibrium dissociationconstants (K_(D)) and dissociative half-lives (t_(1/2)) were calculatedfrom the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2)(min)=ln 2/(60*k_(d)). A set of samples that displayed decreased bindingat pH 6.0 as compared to that at pH 7.4 (pH sensitive) as well as a setof control samples that displayed no significant rate changes betweenthe pH 7.4 and pH 6.0 (pH insensitive controls) were selected to beproduced clonally. FIG. 10 depicts comparison of the number of totalantigen positives and the number of antigen positives displayingpH-sensitive antigen binding from HULC and WT mice.

Among the antigen positives, 18 and 7 clones isolated from twoheterozygous HULC1927 mice and two HULC1930 respectively, and 1 clonefrom the WT mouse, were made monoclonal. Supernatants of the monoclonalhybridomas were subject to neutral and low pH antigen dissociation rate(off-rate) analysis and cell pellets were used for light chain variabledomain DNA sequencing.

Example 3.4 Sequencing and Somatic Hypermutations in CDR3 Region ofHuman Vκ1-39Jκ5-Based Histidine Universal Light Chain Mice

Cell pellets from monoclonal hybridomas from HULC and WT mice were usedfor light chain variable domain DNA sequencing. From the 26 clones mademonoclonal (see Example 3.3 above) and subjected to sequencing, 15 wereconfirmed as using either a HULC or WT mouse light chain (MM and NN, seeTable 4). 14 clones were derived from HULC heterozygous mice (1927 or1930 mice) and 1 was derived from a WT mouse (00, see Table 4).

From the 14 antigen positive samples derived from HULC heterozygousmice, 12 of the monoclonal antibodies utilized their corresponding HULClight chain, while 2 utilized a WT mouse light chain. All but one of theHULC utilizing antibodies retained all of the introduced histidinemutations as shown in Table 3 (italicized antibody). Sequencing of cloneAA produced 2 different HULC sequences, which is reflected by twoentries in Table 3.

TABLE 3 Number of conserved histidine insertions and somatichypermutations in light chain sequences from clones utilizing the HULClight chain Light Chain Sequences from mice utilizing HULC # Somatic #Conserved His # Somatic Hyper- Mouse Mutations in Hypermutationsmutations Clone Name Strain CDR3 in Framework in CDRs AA 1927 4 3 0(Sequence 1) AA 1927 4 1 1 (Sequence 2) BB 1927 4 3 3 CC 1927 4 0 0 DD1927 3 1 1 EE 1927 4 2 2 FF 1927 4 0 1 GG 1927 4 1 1 HH 1927 4 2 0 II1930 3 1 1 JJ 1930 3 4 5 KK 1930 3 1 2 LL 1930 3 1 0

Example 3.5 pH-Dependent Binding of Monoclonal Antibodies Generated inHuman Vκ1-39Jκ5-Based Histidine Universal Light Chain Mice

In order to further assess the pH-dependent binding characteristics ofthe monoclonal antibodies isolated from HULC and WT mice, bindingexperiments were carried out in which the antibody/antigen associationphase was observed at neutral pH and the antibody/antigen dissociationphase was observed at either neutral or acidic pHs.

A Biacore CM4 sensor chip was derivatized with a polyclonal rabbitanti-mouse Fc antibody. Monoclonal antibody supernatants were capturedonto the anti-mouse Fc sensor surface. Two concentrations, 50 nM (induplicate) and 16.7 nM, of the immunogen were injected over themonoclonal antibody captured surface at a flow rate of 30 μl/min.Antibody-antigen association was monitored at pH 7.4 for 4 minutes andthen the dissociation of antigen from the captured monoclonal antibodywas monitored for 15 minutes at either pH 7.4 or 6.0. Dissociation(k_(d)) rate constants were determined by processing and fitting thedata using Scrubber version 2.0 curve fitting software and are shown inTable 4. Dissociative half-lives (t_(1/2)) were calculated from thedissociation rate constants as: t_(1/2) (min)=(ln 2/k_(d))/60, and areshown in Table 4. Sensorgrams depicting the association/dissociationcharacteristics of several antibodies listed in Table 4 under thevarious pH conditions are shown graphically in FIG. 11. The individuallines in each graph represent the binding responses at differentconcentrations of the respective antibodies. All experiments werecarried out at 25° C. Dissociative half-life values (t½) are noted abovethe respective sensorgrams. Response is measured in RU.

TABLE 4 Dissociation (k_(d)) rate constants and dissociative half-lives(t_(1/2)) of monoclonal HULC or WT antibodies binding to their immunogenat neutral and low pH. pH 7.4 Association/pH 7.4 pH 7.4 Association/pH6.0 Dissociation Dissociation 50 nM 50 nM pH Light neutral immunogen lowimmunogen 6.0/pH7.4 Clone chain mAb bound t_(1/2) mab bound t_(1/2)ratio Name used capture (RU) k_(d) (1/s) (min) capture (RU) k_(d) (1/s)(min) k_(d) t_(1/2) AA HULC 129 70 5.60E−05 206 122 73 2.18E−04 53 3.90.3 (1927) BB HULC 350 165 6.00E−04 19 378 185 2.20E−03 5 3.7 0.3 (1927)CC HULC 611 251 2.03E−04 57 545 226 6.68E−03 2 33.0 0.03 (1927) DD HULC182 75 3.55E−04 33 168 74 6.44E−04 18 1.8 0.6 (1927) HH HULC 268 921.36E−04 85 251 91 5.39E−04 21 4.0 0.3 (1927) GG HULC 353 110 2.78E−0442 328 102 8.97E−04 13 3.2 0.3 (1927) FF HULC 334 202 4.79E−05 241 364220 6.90E−05 167 1.4 0.7 (1927) EE HULC 339 124 5.08E−04 23 299 1204.66E−04 25 0.9 1.1 (1927) II HULC 387 174 1.22E−04 95 334 147 2.14E−0454 1.8 0.6 (1930) JJ HULC 363 14 9.83E−04 12 333 12 5.30E−04 22 0.5 1.9(1930) KK HULC 490 303 7.41E−05 156 484 295 1.29E−04 90 1.7 0.6 (1930)LL HULC 636 41 3.09E−04 37 597 36 5.77E−04 20 1.9 0.5 (1930) MM* WT 2456 NA NA 203 6 NA NA NA NA (from 1927 mouse) NN WT 394 231 5.26E−04 22378 231 9.35E−04 12 1.8 0.6 (from 1927 mouse) OO WT 413 89 2.94E−04 39400 83 3.57E−04 32 1.2 0.8 *k_(d) and t_(1/2) values could not bedetermined due to low antigen binding signal

Example 4 Engineering of Genetically Modified Mouse Comprising aHistidine-Substituted Human Vκ3-20Jκ1 Universal Light Chain

A mouse comprising a common Vκ3-20Jκ1 light chain was generated asdescribed in, e.g., U.S. patent application Ser. Nos. 13/022,759,13/093,156, 13/412,936, and 13/488,628 (Publication Nos. 2011/0195454,2012/0021409, 2012/0192300, and 2013/0045492, respectively), and inExample 1 above. The amino acid sequence of the germline universalVκ3-20Jκ1 light chain variable domain is set forth in SEQ ID NO:59.

Histidine substitutions were introduced into the Vκ3-20Jκ1 universallight chain targeting vector and mice generated from the same using asimilar strategy to the one described above in Example 3 for Vκ1-39Jκ5histidine modified universal light chain mice (HULC 1927 and 1930).

Briefly, the strategy for generating a histidine-modified Vκ-3-20Jκ1universal light chain targeting vector is summarized in FIGS. 14A-14D. Aplasmid used for generating a targeting vector for common (universal)light chain mouse (“ULC mouse,” described in, e.g., US2011/0195454A1),containing pBS+FRT-Ub-Hyg-FRT+mouse Vκ3-7 leader+human Vκ3-20Jκ1 wasmodified by site directed mutagenesis (QuickChange Lightning Kit) toreplace Q105, Q106, Y107 and S109 or Q105, Q106 and S109 (see alignmentin FIG. 12) with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 13 (See FIG. 14A forthis engineering step). Resultant vectors (H105/106/107/109 andH105/106/109) were modified further and ligated into a vector comprisingmouse Igκ constant region, mouse enhancers, a mouse 3′ homology arm anda SPEC cassette (FIG. 14B). Further modification involved ligation intoa vector carrying 5′ mouse arm and comprising Frt-UB-NEO-Frt cassette(FIG. 14B). Resultant targeting vectors were electroporated into EScells comprising deletion of the mouse Igκ variable locus (comprising κvariable and joining gene segments) (FIGS. 14C-14D).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ3-20κJ1 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 5 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 14C-14D.

TABLE 5 Primers and Probes Used for ES Cell Screening Probe Name AssayProbe Sequence 5′ Primer 3′ Primer Neo GOA TGGGCACAACAGAC GGTGGAGAGGGAACACGGCGG AATCGGCTG CTATTCGGC CATCAG (SEQ ID NO: 38) (SEQ ID NO: 39)(SEQ ID NO: 40) ULC-m1 GOA CCATTATGATGCTCC AGGTGAGGGT TGACAAATGCCCATGCCTCTCTGTTC ACAGATAAGTG TAATTATAGTGAT (SEQ ID NO: 41) TTATGAG CA(SEQ ID NO:42) (SEQ ID NO: 43) 1635h2 GOA AAAGAGCCACCCTCT TCCAGGCACCCAAGTAGCTGCTGCTA (Vκ3-20Jκ1 CCTGCAGGG TGTCTTTG ACACTCTGACT specific)(SEQ ID NO: 65) (SEQ ID NO: 66) (SEQ ID NO: 67) mlgKd2 RetentionGGCCACATTCCATGG GCAAACAAAAACCAC CTGTTCCTCTAAAAC GTTC TGGCCTGGACTCCACAGTAA (SEQ ID NO: 47) (SEQ ID NO: 48) ATGGAAA (SEQ ID NO: 49)mlgKpl5 Retention GGGCACTGGATACGA CACAGCTTGTGCAGC AGAAGAAGCCTGTACTGTATGG CTCC TACAGCATCCGTTTT (SEQ ID NO: 50) (SEQ ID NO: 51) ACAGTCA(SEQ ID NO: 52)

The NEO selection cassette introduced by the targeting constructs isdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 14C and 14D). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above are used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence are made from the targeted ES cellsdescribed above.

Pups are genotyped and pups heterozygous for the engineeredhistidine-modified human light chain are selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H105/106/109;“6183”) or four (H105/105/108/111; “6181”) histidine modifications arelisted in Table 6 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 6 Primers and Probes Used for Genotyping Probe Name AssayProbe Sequence 5′ Primer 3′ Primer hVI494-1 GOA 6181 (4 CTGTCATCACCATGGGCAGACTGGAGCCTG CCGAACGTCCAAGGT His) mouse- AAGATTTT GAGTG specific(SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70) hVI495-1 GOA 6183 (3TACTGTCATCACTAT GCAGACTGGAGCCTG CCGAACGTCCAAGGT His) mouse- GG AAGATTTGAGTG specific (SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 73)

Mice are immunized with antigen of interest and tested for ability togenerate antibodies with pH-dependent binding.

Example 5 Breeding of Mice Comprising a Histidine-Substituted HumanUniversal Light Chains (HULC)

This Example describes several other genetically modified mouse strainsthat can be bred to any one of the human HULC mice described herein tocreate multiple genetically modified mouse strains harboring multiplegenetically modified immunoglobulin loci.

Endogenous Igλ Knockout (KO).

To optimize the usage of the engineered light chain locus, any one ofthe HULC animals described above (e.g., comprising Vκ1-39Jκ5 orVκ3-20Jκ1 histidine-substituted universal light chain) may be bred toanother mouse containing a deletion in the endogenous λ, light chainlocus. In this manner, the progeny obtained will express, as their onlylight chain, the rearranged histidine-substituted human germline lightchain region as described in Examples 3 and 4 above. Breeding isperformed by standard techniques recognized in the art and,alternatively, by a commercial breeder (e.g., The Jackson Laboratory).Mouse strains bearing an engineered histidine-substituted light chainlocus and a deletion of the endogenous λ, light chain locus are screenedfor presence of the unique light chain region and absence of endogenousmouse λ, light chains.

Humanized Endogenous Heavy Chain Locus.

Mice bearing an engineered human germline light chain locus (HULC mice)are bred with mice that contain a replacement of the endogenous mouseheavy chain variable gene locus with the human heavy chain variable genelocus (see U.S. Pat. No. 6,596,541 and U.S. Pat. No. 8,502,018; theVELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.). The VELOCIMMUNE®mouse comprises a genome comprising human heavy chain variable regionsoperably linked to endogenous mouse constant region loci such that themouse produces antibodies comprising a human heavy chain variable domainand a mouse heavy chain constant region in response to antigenicstimulation.

Mice bearing a replacement of the endogenous mouse heavy chain variableregion locus with the human heavy chain variable region locus and ahistidine-substituted single rearranged human light chain variableregion at the endogenous κ light chain locus are obtained. Reversechimeric antibodies containing somatically mutated heavy chains (humanheavy chain variable domain and mouse C_(H)) with ahistidine-substituted single human light chain (HULC, human light chainvariable domain and mouse C_(L)) are obtained upon immunization with anantigen of interest. pH-dependent human antibodies generated in suchmice are identified using antibody isolation and screening methods knownin the art or described above. Variable light and heavy chain regionnucleotide sequences of B cells expressing the antibodies, e.g.,pH-sensitive antibodies, are identified, and fully human antibodies aremade by fusion of the variable heavy and light chain region nucleotidesequences to human C_(H) and C_(L) nucleotide sequences, respectively,in a suitable expression system.

Example 6 pH-Dependent Binding of Antibodies Generated in MiceComprising Histidine-Substituted Human Universal Light Chains (HULC) andHuman Heavy Chain Variable Domains

Mice bearing engineered Vκ1-39/Jκ5 ULC (1633) or Vκ1-39/Jκ5 comprisingeither 4 histidine substitutions (HULC 1927) or 3 histidinesubstitutions (HULC 1930) were bred to mice comprising a replacement ofthe endogenous mouse heavy chain variable region locus with the humanvariable region locus (see U.S. Pat. No. 6,596,541 and U.S. Pat. No.8,502,018, the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.).Mice homozygous for both human heavy chain variable region locus andeither engineered ULC (labeled as “ULC 1633ho/human heavy ho”),Vκ1-39/Jκ5 comprising 4 histidine substitutions (labeled as “HULC1927ho/human heavy ho”), or Vκ1-39/Jκ5 comprising 3 histidinesubstitutions (labeled as “HULC 1930ho/human heavy ho”) were obtained.

Subsequently, the genetically engineered mice homozygous formodifications at both light and heavy chain loci described above wereused for immunization with cytokine receptor (“Antigen B”). Three ULC1633ho/human heavy ho, seven HULC 1927ho/human heavy ho, and six HULC1930ho/human heavy ho mice were used for immunization.

The mice were terminated and splenocytes were harvested. Red blood cellswere removed by lysis followed by pelleting the harvested splenocytes.Resuspended splenocytes were incubated with a cocktail of reagents thatcan allow identification and isolation of antigen positive B cells.Cells were analyzed by flow cytometery. Each IgG positive, IgM negative,and antigen positive B cell was sorted and plated into a separate wellon a 384 well plate. Individual B cells were subjected to PCR to amplifyantigen-specific heavy and light chain variable domains. The amplifiedheavy and light chain variable domains were cloned into antibody vectorscontaining human IgG1 heavy chain constant region and light chainconstant region, respectively. Purified recombinant plasmids havingheavy and light chain variable sequence from the same B cell wereco-transfected and expressed in a CHO host cell line.

Expressed antibodies were subjected to affinity screening using areal-time surface plasmon resonance biosensor (Biacore 4000). Kineticbinding parameters (e.g., k_(a), k_(d), K_(D), t_(1/2), etc.) forantibody binding to the immunogen at neutral pH (pH 7.4) and at acidicpH (pH 6.0) were recorded. A Biacore CM4 sensor chip was derivatizedwith a monoclonal mouse anti-human Fc antibody to capture antibodiesfrom the supernatant. A single concentration (100 nM) of immunogen wasthen injected over the antibody-captured surface at a flow rate of 30μl/min. Antibody-antigen association was monitored for 1.5 minutes andthen the dissociation of antigen from the captured antibody wasmonitored for 2.5 minutes. Kinetic association (k_(a)) and dissociation(k_(d)) rate constants were determined by processing and fitting thedata to a 1:1 binding with a mass transport model using Biacore 4000Evaluation software version 1.0. Equilibrium dissociation constants(K_(D)) and dissociative half-lives (t_(1/2)) were calculated from thekinetic rate constants as: K_(D) (M)=k_(d) k_(a); and t_(1/2) (min)=ln2/(60*k_(d)).

Biacore binders were defined as any antibodies that have a measurableK_(D) pH-dependent binder, in this experiment, was defined as anyantibody that has a ratio of t_(1/2) at pH 7.4 to t_(1/2) at pH 6.0 ofgreater than about 2.

As shown in Table 7 below, there was a 2-3 fold increase in percentageof antibodies that displayed pH-dependent antigen binding in the 1927and 1930 HULC mice in comparison to 1633 ULC mice.

TABLE 7 Percentage of pH-Dependent Antibodies Generated in HULC Mice pHBiacore dependent % pH Mouse Strain Binders binding dependent ULC1633ho/human heavy ho 115 10 8% HULC 1930ho/human heavy ho 205 49 24%HULC 1927ho/human heavy ho 34 7 21%

Example 7 Generation and Analysis of Mice Comprising Two Human VSegments Example 7.1 Construction of Targeting Vector for Generation ofMice That Comprise Two Human V Segments

Two engineered light chain loci containing two human Vκ gene segments(e.g., a human Vκ1-39 and human Vκ3-20 gene segment) were constructed(FIG. 16B). One engineered light chain locus contained two human Vκ genesegments and five human Jκ gene segments in unrearranged configuration(DLC-5J). The second engineered light chain locus contained two human Vκgene segments and one human Jκ gene segment in unrearrangedconfiguration (DLC-1J). For each of the two additional engineered lightchain loci, the human gene segments were flanked 3′ with recombinationsignal sequences to allow for in vivo rearrangement of the human genesegments in B cells.

Engineering and Generation of DLC-1J Mice.

Engineering steps that result in generation of a light chain locuscomprising two human Vκ gene segments (Vκ1-39 and Vκ3-20) and one humanJκ gene segment (Jκ5), otherwise termed as DLC-1J, are depicted in FIG.17. Specifically, human Vκ1-39 and Vκ3-20 sequences were amplified byPCR from BAC templates (Invitrogen), and together with an amplifiedsequence containing recombination signal sequence (rss) and human Jκ5segment, cloned via a four-way ligation into a plasmid containing aUB-hygromycin selection cassette (FIG. 17A). 5′ and 3′ arms wereattached as depicted in FIGS. 17B and 17C.

The resultant targeting construct is depicted in FIG. 16B (bottomdiagram; DLC-1J), with recombination signal sequences (RSS) in clearovals. Modified BAC DNA clone of the engineered DLC-1 J light chainlocus operably linked to mouse sequences (i.e., upstream and downstreamsequences of the endogenous immunoglobulin κ light chain locus) wasconfirmed by PCR using primers located at sequences within theengineered light chain locus containing the two human Vκ gene segments,followed by electroporation into ES cells comprising deletion of themouse Igκ variable locus (comprising κ variable and joining genesegments) (FIG. 17D) to create a mouse that expresses either of the twohuman Vκ gene segments. Positive ES cell clones that contained theengineered DLC-1J light chain locus was confirmed by TAQMAN™ screeningand karyotyping using probes specific for the engineered DLC-1J lightchain locus. Sequences of primers and probes used for ES cell screeningof DLC-1J ES cells are depicted in Table 8 below and are included inSequence Listing.

TABLE 8 Primers and Probes Used for ES Cell Screening Probe Assay/typeLocation Name of probe detected Probe Sequence Forward PrimerReverse Primer 1633h2 GOA/ Vκ1-39 ATCAGCAGAAACCAG GGGCAAGTCAGAGCATGCAAACTGGATGCA TAQMAN ™ GGAAAGCCCCT TTAGCA GCATAG (SEQ ID NO: 44)(SEQ ID NO: 45) (SEQ ID NO: 46) 1635h2 GOA/ Vκ3-20 AAAGAGCCACCCTCTTCCAGGCACCCTGTC AAGTAGCTGCTGCTA TAQMAN ™ CCTGCAGGG TTTG ACACTCTGACT(SEQ ID NO: 65) (SEQ ID NO: 66) (SEQ ID NO: 67) Neo GOA neoTGGGCACAACAGACA GGTGGAGAGGCTATT GAACACGGCGGCATC ATCGGCTG CGGC AG(SEQ ID NO: 38) (SEQ ID NO: 39) (SEQ ID NO: 40) Jxn GOA/BHQ1 1-39/3-20TCTTTTGCCCCGGAT GGGAGGCTCCTCTGA GTCCAGTCACTCGGT 1-39/3-20 BamHI CCGATCAGACTCTAAG TGCTAT junction (SEQ ID NO: 84; (SEQ ID NO: 85) (SEQ ID NO: 86)restriction site bolded)

Confirmed ES cell clones were then used to implant female mice to giverise to a litter of pups comprising DLC-1J light chain locus andexpressing a human light chain variable domain fused with a mouse Cκdomain. Sequences of primers and probes used for genotyping of the pupsare listed in Table 8 above. The sequence through the engineered DLC-1Jlocus, including about 100 nucleotides of mouse sequence upstream anddownstream of the inserted engineered sequence is presented in FIG. 18and set forth in SEQ ID NO:82.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP in order to remove the FRTedneomycin cassette introduced by the targeting construct (see FIG. 17E).Optionally, the neomycin cassette is removed by breeding to mice thatexpress FLP recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theneomycin cassette is retained in the mice.

Engineering and Generation of DLC-5J Mice.

To generate a light chain locus comprising two human Vκ gene segments(Vκ1-39 and Vκ3-20) and five human Jκ gene segments (Jκ1, Jκ2, Jκ3, Jκ4,and Jκ5), otherwise termed as DLC-5J, a 2000 base pair amplifiedsequence comprising all 5 human Jκ's was ligated into a vectorcomprising two human Vκ gene segments and one human Jκ, depicted in FIG.17B (middle) (see FIG. 19A). Subsequent engineering steps involvedattachment of 3′ and 5′ arms as depicted in FIG. 19B.

The resultant targeting construct is depicted in FIG. 16B (top diagram;DLC-5J), with recombination signal sequences (RSS) in clear ovals.Modified BAC DNA clone the engineered DLC-5J light chain locus operablylinked to mouse sequences (i.e., upstream and downstream sequences ofthe endogenous immunoglobulin κ light chain locus) was confirmed by PCRusing primers located at sequences within the engineered light chainlocus containing the two human Vκ gene segments, followed byelectroporation into ES cells comprising deletion of the mouse Igκvariable locus (comprising κ variable and joining gene segments) (FIG.19C) to create a mouse that expresses either of the two human Vκ genesegments. Positive ES cell clones that contained the engineered DLC-5Jlight chain locus was confirmed by TAQMAN™ screening and karyotypingusing probes specific for the engineered DLC-5J light chain locus.Sequences of primers and probes used for ES cell screening of DLC-5J EScells are depicted in Table 9 below and are included in SequenceListing.

TABLE 9 Primers and Probes Used for ES Cell Screening Assay/typeLocation Probe Name of probe detected Probe Sequence Forward PrimerReverse Primer 1633h2 GOA/ Vκ1-39 ATCAGCAGAAACCAG GGGCAAGTCAGAGCATGCAAACTGGATGCA TAQMAN ™ GGAAAGCCCCT TTAGCA GCATAG (SEQ ID NO: 44)(SEQ ID NO: 45) (SEQ ID NO: 46) 1635h2 GOA/ Vκ3-20 AAAGAGCCACCCTCTTCCAGGCACCCTGTC AAGTAGCTGCTGCTA TAQMAN ™ CCTGCAGGG TTTG ACACTCTGACT(SEQ ID NO: 65) (SEQ ID NO: 66) (SEQ ID NO: 67) Neo GOA neoTGGGCACAACAGACA GGTGGAGAGGCTATT GAACACGGCGGCATC ATCGGCTG CGGC AG(SEQ ID NO: 38) (SEQ ID NO: 39) (SEQ ID NO: 40) Jxn GOA/BHQ1 1-39/3-20TCTTTTGCCCCGGAT GGGAGGCTCCTCTGA GTCCAGTCACTCGGT 1-39/3-20 BamHI CCGATCAGACTCTAAG TGCTAT junction (SEQ ID NO: 84; (SEQ ID NO: 85) (SEQ ID NO: 86)restriction site bolded) Jxn GOA/BHQ1 3-20/Jk1-5 CTTCAACTGTGGCGTACGCAGATGTAGCCA CAGCTGCTGAAGCTC 3-20/Jk1-5 BsiWI ACGCACC AACCCT AACTCjunction (SEQ ID NO: 87, (SEQ ID NO: 88) (SEQ ID NO: 89) restrictionsite bolded)

Confirmed ES cell clone was then used to implant female mice to giverise to a litter of pups comprising DLC-5J light chain locus andexpressing a human light chain variable domain fused with a mouse Cκdomain. Sequences of primers and probes used for genotyping of the pupsare listed in Table 9 above. The sequence through the engineered DLC-5Jlocus, including about 100 nucleotides of mouse sequence upstream anddownstream of the inserted engineered sequence is presented in FIG. 20and set forth in SEQ ID NO:83.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP in order to remove the FRTedneomycin cassette introduced by the targeting construct (see FIG. 19D).Optionally, the neomycin cassette is removed by breeding to mice thatexpress FLP recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theneomycin cassette is retained in the mice.

Example 7.2 Characterization of Mice that Comprise Two Human V Segments

Flow Cytometry.

B cell populations and B cell development in DLC mice were validated byflow cytometry analysis of splenocyte and bone marrow preparations. Cellsuspensions from mice homozygous for two human Vκ gene segments and fivehuman Jκ gene segments (n=4), mice homozygous for two human Vκ genesegments and one human Jκ gene segment (n=4), and wild type mice (n=4)were made using standard methods and stained with fluorescently labeledantibodies.

Briefly, 1×10⁶ cells were incubated with anti-mouse CD16/CD32 (clone2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling withthe following antibody cocktail for 30 minutes on ice: APC-H7 conjugatedanti-mouse CD19 (clone 1D3, BD Pharmigen), Pacific Blue conjugatedanti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ(clone 187.1, BD Pharmigen) or anti-mouse CD43 (clone 1B11, BioLegend),PE conjugated anti-mouse Igλ, (clone RML-42, BioLegend) or anti-mousec-kit (clone 2B8, BioLegend), PerCP-Cy5.5 conjugated anti-mouse IgD(BioLegend), PE-Cy7 conjugated anti-mouse IgM (clone II/41,eBioscience), APC conjugated anti-mouse B220 (clone RA3-6B2,eBioscience). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on an LSRII flow cytometerand analyzed with FlowJo (Tree Star, Inc.). Gating: total B cells(CD19⁺CD3⁻), Igκ⁺ B cells (Igκ⁺Igλ⁻CD19⁺CD3⁻), Igλ⁺ B cells(Igκ⁻Igλ⁺CD19⁺CD3⁻). Results for the bone marrow compartment are shownin FIG. 21A-23B. Results for the splenic compartment are shown in FIG.24A-FIG. 27.

As shown in this Example, DLC-5J mice demonstrate normal B cellpopulations within the splenic and bone marrow compartments (FIG.21A-27). DLC-5J mice demonstrated immature, mature and pre/pro B cellpopulations within the bone marrow compartment that are substantiallythe same as observed in wild-type litter mates. In fact, the DLC-5Jlocus was capable of competing with the endogenous λ light chain locusto yield a κ:λ, ratio that is substantially the same as that observed inwild-type mice (FIG. 25B). Also, DLC-5J mice demonstrate a normalperipheral B cell development as progression of B cells through variousstages in the splenic compartment (e.g., immature, mature, T1, T2 T3,marginal zone precursor, marginal zone, follicular-I, follicular-II,etc.) occurs in a manner substantially the same as observed in wild typemice (FIG. 26A-27). In contrast, DLC-1J mice demonstrated a loweroverall number of B cells and an increased λ light chain usage ascompared to the engineered κ light chain (data not shown).

Dual Light Chain Expression.

Expression of both human Vκ gene segments was analyzed in homozygousmice using a quantitative PCR assay. Briefly, CD19⁺ B cells werepurified from bone marrow and whole spleens of wild type mice, micehomozygous for a replacement of the mouse heavy chain and κ light chainvariable loci with corresponding human heavy chain and κ light chainvariable region loci (Hκ), as well as mice homozygous for an engineeredκ light chain loci containing two human Vκ gene segments and either fivehuman Jκ gene segments (DLC-5J) or one human Jκ gene segment (DLC-1J).Relative expression was normalized to expression of mouse Cκ region (n=3to 5 mice per group). Results are shown in FIG. 28 and FIG. 29.

Expression of light chains containing a rearranged human Vκ3-20 or humanVκ1-39 gene segment were detected in both the bone marrow and spleen ofDLC-5J and DLC-1J mice (FIG. 28 and FIG. 29). In the bone marrowcompartment, expression of both human Vκ/3-20-derived and humanVκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to mice comprising a replacement ofmouse Vκ and Jκ gene segment with corresponding human Vκ and Jκ genesegments (Hκ; FIG. 28). Human Vκ3-20-derived light chain expression wasobserved at about six-fold (DLC-5J) to fifteen-fold (DLC-1 J) higherthan in Hκ mice. DLC-1 J mice demonstrated about two-fold higherexpression of human Vκ3-20-derived light chains over DLC-5J mice in thebone marrow compartment. Human Vκ1-39-derived light chain expression wasobserved at about six-fold (DLC-5J) to thirteen-fold (DLC-1J) higherthan in Hκ mice. DLC-1J mice demonstrated about two-fold higherexpression of human Vκ1-39-derived light chains over DLC-5J mice in thebone marrow compartment.

In the splenic compartment, expression of both human Vκ3-20-derived andhuman Vκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to Hκ mice (FIG. 29). HumanVκ3-20-derived light chain expression was observed at about four-fold(DLC-5J) and eight-fold (DLC-1 J) higher than in Hκ mice. DLC-1 J micedemonstrated about two-fold higher expression of human Vκ3-20-derivedlight chains over DLC-5J mice in the splenic compartment. HumanVκ1-39-derived light chain expression was observed at about four-fold(DLC-5J) to five-fold (DLC-1 J) higher than in Hκ mice. DLC-1 J micedemonstrated similar expression of human Vκ1-39-derived light chains ascompared to DLC-5J mice in the splenic compartment.

Human V_(L)/J_(L) Usage in DLC-5J Mice.

Mice homozygous for two unrearranged human Vκ gene segments and fiveunrearranged human Jκ gene segments (DLC-5J) were analyzed for humanVκ/Jκ gene segment usage in splenic B cells by reverse-transcriptasepolymerase chain reaction (RT-PCR).

Briefly, spleens from homozygous DLC-5J (n=3) and wild type (n=2) micewere harvested and meshed in 10 mL of RPMI 1640 (Sigma) containing 10%heat-inactivated fetal bovine serum using frosted glass slides to createsingle cell suspensions. Splenocytes were pelleted with a centrifuge(1200 rpm for five minutes) and red blood cells were lysed in 5 mL ofACK lysing buffer (GIBCO) for three minutes. Splenocytes were dilutedwith PBS (Irvine Scientific), filtered with a 0.7 μm cell strainer andcentrifuged again to pellet cells, which was followed by resuspension in1 mL of PBS.

RNA was isolated from pelleted splenocytes using AllPrep DNA/RNA minikit (Qiagen) according to manufacturer's specifications. RT-PCR wasperformed on splenocyte RNA using 5′ RACE (Rapid Amplification of cDNAends) System with primers specific for the mouse Cκ gene according tomanufacturer's specifications (Invitrogen). The primers specific for themouse Cκ gene were 3′ mIgκC RACE1 (AAGAAGCACA CGACTGAGGC AC; SEQ ID NO:90) and mIgκC3′-1 (CTCACTGGAT GGTGGGAAGA TGGA; SEQ ID NO: 91). PCRproducts were gel-purified and cloned into pCR®2.1-TOPO® vector (TOPO®TA Cloning® Kit, Invitrogen) and sequenced with M13 Forward (GTAAAACGACGGCCAG; SEQ ID NO: 92) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:93) primers located within the vector at locations flanking the cloningsite. Ten clones from each spleen sample were sequenced. Sequences werecompared to the mouse and human immunoglobulin sets from theIMGT/V-QUEST reference directory sets to determine Vκ/Jκ usage. Table 10sets forth the Vκ/Jκ combinations for selected clones observed in RT-PCRclones from each splenocyte sample. Table 11 sets forth the amino acidsequence of the human Vκ/human Jκ and human Jκ/mouse Cκ junctions ofselected RT-PCR clones from DLC-5J homozygous mice. Lower case lettersindicate mutations in the amino acid sequence of the variable region ornon-template additions resulting from N and/or P additions duringrecombination.

As shown in this Example, mice homozygous for two unrearranged human Vκgene segments and five unrearranged human Jκ gene segments (DLC-5J)operably linked to the mouse Cκ ene are able to productively recombineboth human Vκ gene segments to multiple human Jκ gene segments toproduce a limited immunoglobulin light chain repertoire. Among therearrangements in DLC-5J homozygous mice shown in Table 10, unique humanVκ/Jκ rearrangements were observed for Vκ1-39/Jκ2 (1), Vκ1-39/Jκ3 (1),Vκ3-20/Jκ1 (7), Vκ3-20/Jκ2 (4) and Vκ3-20/Jκ3 (1). Further, such uniquerearrangements demonstrated junctional diversity through the presence ofunique amino acids within the CDR3 region of the light chain (Table 11)resulting from either mutation and/or the recombination of the human Vκand Jκ gene segments during development. All the rearrangements showedfunctional read through into mouse Cκ (Table 11).

Taken together, these data demonstrate that mice engineered to present achoice of no more than two human V_(L) gene segments, both of which arecapable of rearranging (e.g., with one or more and, in some embodiments,up to five human J_(L) gene segments) and encoding a human V_(L) domainof an immunoglobulin light chain have B cell numbers and developmentthat is nearly wild-type in all aspects. Such mice produce a collectionof antibodies having immunoglobulin light chains that have one of twopossible human V_(L) gene segments present in the collection. Thiscollection of antibodies is produced by the mouse in response to antigenchallenge and are associated with a diversity of reverse chimeric (humanvariable/mouse constant) heavy chains.

TABLE 10 Vκ/Jκ Combinations Observed in Splenocyte Samples Mouse ID No.Genotype Clone Vκ/Jκ Combination 1089451 DLC-5J 1-2 1-39/3 1-4 3-20/21-7 3-20/1 1-8 3-20/2 1089452 DLC-5J 2-2 3-20/1 2-3 3-20/1 2-6 3-20/22-8 3-20/2 2-9 3-20/1 2-10 1-39/2 1092594 DLC-5J 3-1 3-20/1 3-2 3-20/13-4 3-20/1 3-6 3-20/3 3-9 3-20/2 1092587 WT 1-1 19-93/1 1-2 6-25/1 1-34-91/5 1-5 3-10/4 1-6 4-86/4 1-8 19-93/1 1-10 19-93/2 1092591 WT 2-119-93/1 2-3 6-20/5 2-4 6-25/5 2-5 1-117/1 2-6 8-30/1 2-7 8-19/2 2-88-30/1 2-10 1-117/1

TABLE 11 Amino Acid Sequences of the Human Vκ/Human Jκand Human Jκ/Mouse Cκ Junctions from DLC-5J Homozygous MiceSequence of hVκ/hJκ/mCκ Junction SEQ Clone Vic/JK(CDR3 underlined, mlgκC italics) ID NO:  2-10 1-39/2QPEDFATYYCQQSYSTPYTFGQGTKLEIKRADAAPTVSI 94 1-2 1-39/3QPEDFATYYCQQSYSTPFTFGPGTKVDIKRADAAPTVSI 95 1-7 3-20/1EPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 96 2-2 3-20/1EPEDFAVYYCQQYGSSrTFGQGTKVEIKRADAAPTVSI 97 2-3 3-20/1EPEDFAVYYCQQYGSSPWTFGQGTKVEIKRADAAPTVSI 98 2-9 3-20/1dPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 99 3-1 3-20/1EPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 100  3-2 3-20/1EPEDFAVYYCQQYGSSPWTFGQGTKVEIKRADAAPTVSI 101  3-4 3-20/1EPEDFAVYYCQQYGSSPPTFGQGTKVEIKRADAAPTVSI 102  3-9 3-20/2EPEDFAVYYCQQYGSSPYTFGQGTKLEIKRADAAPTVSI 103  3-6 3-20/3EPEDFAVYYCQQYGSSiFTFGPGTKVDIKRADAAPTVSI 104 

Example 8 Generation and Characterization of Mice Comprising TwoHistidine-Substituted Human Light Chains Example 8.1 Engineering andGeneration of Mice Comprising Two V Kappa Segments Each Containing FourHistidine Substitutions

Histidine substitutions were introduced into the dual light chain locusas described above for Vκ1-39 and Vκ3-20 ULC mice. Briefly, the DLCsequence depicted in FIG. 19A (bottom) was subjected to site-directedmutagenesis, first modifying the Vκ1-39 sequence, and subsequentlymodifying the Vκ3-20 sequence, using primers depicted in FIG. 30. Theresultant dual light chain sequence contained Vκ1-39 segment withhistidines introduced into the germline sequence at positions 105, 106,108, and 111, Vκ3-20 segment with histidines introduced into thegermline sequence at positions 105, 106, 107, and 109, as well as allfive Jκ segments (Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5). A subsequent engineeringstep involved attachment of a 5′ arm carrying an FRT-UB-NEO-FRTcassette, and a 3′ arm carrying a mouse Igκ enhancers and constantregion. This targeting vector was electroporated into ES cellscomprising deletion of the mouse Igκ variable locus (comprising κvariable and joining gene segments), as depicted in FIG. 31A(recombination signal sequences, RSS, are omitted in this figure).Targeted ES cells were screened by a modification of allele assay asdescribed above, using primers and probes that detected the regionsdescribed above in Tables 1, 5, 8, and 9 (specifically, 1633h2, 1635h2,neo, Jxn 1-39/3-20, mIgKd2, and mIgKp15), as well as two additional setsof primers and probes listed in Table 12 below. The sequences of thesetwo additional sets of primers and probes are included in the SequenceListing.

TABLE 12 Primers and Probes Used for ES Cell Screening Probe Assay/typeLocation Name of probe detected Probe Sequence Forward PrimerReverse Primer hVI492 GOA/ MAID 6185 AACTTACTACTGTCAC CAGCAGTCTGCAACCTGGCTCGTCCTCACACA 1-39 FAM-BHQ+ (4 HIS-1-39 CA GAA TC specific)(SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 112) hVI492 GOA/ MAID 61853-20 FAM-BHQ+ (4 HIS-3-20 TTACTGTCACCATCAT GCAGACTGGAGCCTGAAAGCTGAATCACTGTG specific) G AGA GGAGGTG (SEQ ID NO: 114)(SEQ ID NO: 115 (SEQ ID NO: 116)

Confirmed ES cell clone is then used to implant female mice to give riseto a litter of pups comprising DLC-5J light chain locus with fourhistidine modifications at each of the two present V_(L) segmentsequences, and expressing a human light chain variable domain fused witha mouse Cκ domain. Some of the same sequences as used for ES cellscreening are also used for genotyping of pups.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP (e.g., FLPo) in order to remove theFRTed neomycin cassette introduced by the targeting construct (see FIG.31B, RSS are omitted in this figure). Optionally, the neomycin cassetteis removed by breeding to mice that express FLP recombinase (e.g., U.S.Pat. No. 6,774,279). Optionally, the neomycin cassette is retained inthe mice.

Example 8.2 Engineering and Generation of Mice Comprising Two V KappaSegments Each Containing Three Histidine Substitutions

Three histidine substitutions were introduced into each Vκ1-39 andVκ3-20 of the dual light chain mice. Briefly, the DLC sequence depictedin FIG. 19A (bottom) was subjected to site-directed mutagenesis, firstmodifying the Vκ1-39 sequence, and subsequently modifying the Vκ3-20sequence, using primers depicted in FIG. 32. The resultant dual lightchain sequence contained Vκ1-39 segment with histidines introduced intothe germline sequence at positions 106, 108, and 111, Vκ3-20 segmentwith histidines introduced into the germline sequence at positions 105,106, and 109, as well as all five Jκ segments (Jκ1, Jκ2, Jκ3, Jκ4, andJκ5). A subsequent engineering step involved attachment of a 5′ armcarrying an FRT-UB-NEO-FRT cassette, and a 3′ arm carrying a mouse Igκenhancers and constant region. This targeting vector was electroporatedinto ES cells comprising deletion of the mouse Igκ variable locus(comprising κ variable and joining gene segments), as depicted in FIG.33A (RSS are omitted in this figure). Targeted ES cells were screened bya modification of allele assay as described above, using primers andprobes that detected the regions described above in Tables 1, 5, 8, and9 (specifically, 1633h2, 1635h2, neo, Jxn 1-39/3-20, mIgKd2, andmIgKp15), as well as two additional sets of primers and probes listed inTable 13 below. The sequences of these two additional sets of primersand probes are included in the Sequence Listing.

TABLE 13 Primers and Probes Used for ES Cell Screening Probe Assay/typeLocation Name of probe detected Probe Sequence Forward PrimerReverse Primer hVI493 GOA/ MAID 6187 CTTACTACTGTCAACA CAGCAGTCTGCAACCTGGCTCGTCCTCACACA 1-39 FAM-BHQ+ (3 HIS-1-39 TAG GAA TC specific)(SEQ ID NO: 123) (SEQ ID NO: 124) (SEQ ID NO: 125) hVI493 GOA/ MAID 6187TACTGTCACCATTATG GCAGACTGGAGCCTGA AAGCTGAATCACTGTG 3-20 FAM-BHQ+(3 HIS-3-20 G AGA GGAGGTG specific) (SEQ ID NO: 126) (SEQ ID NO: 127)(SEQ ID NO: 128)

Confirmed ES cell clone is then used to implant female mice to give riseto a litter of pups comprising DLC-5J light chain locus with fourhistidine modifications at each of the two present V_(L) segmentsequences, and expressing a human light chain variable domain fused witha mouse Cκ domain. Some of the same sequences as used for ES cellscreening are also used for genotyping of pups.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP (e.g., FLPo) in order to remove theFRTed neomycin cassette introduced by the targeting construct (see FIG.33B, RSS are omitted in this figure). Optionally, the neomycin cassetteis removed by breeding to mice that express FLP recombinase (e.g., U.S.Pat. No. 6,774,279). Optionally, the neomycin cassette is retained inthe mice.

Example 8.3 Breeding of Mice Comprising a Human Histidine-SubstitutedDual Light Chains

Mice bearing an engineered human histidine-substituted dual light chainlocus are bred with mice that contain a deletion of the endogenous λ,light chain locus to generate progeny that expresses, as their onlylight chains, the engineered histidine-substituted light chains derivedfrom the dual light chain locus.

Mice bearing an engineered human histidine-substituted dual light chainlocus are bred with mice that contain a replacement of the endogenousmouse heavy chain variable locus with human heavy chain variable locus(see U.S. Pat. No. 6,596,541 and U.S. Pat. No. 8,502,018; theVELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.).

Similar breedings to the ones described herein are set up for dual lightchain mice described in Example 7 above.

Further details of these breeding methods and the generation of fullyhuman antibodies from the human variable light and heavy chain regionsare described in Example 5 above.

Example 8.4 Detection of Histidine Modifications in Immunoglobulin LightChains Obtained from Mice Comprising Two V Kappa Segments EachContaining Three Histidine Substitutions

V kappa amplicons from splenic B cell mRNA was prepared usingreverse-transcriptase PCR (RT-PCR) and high throughput screening.

Briefly, spleens from five heterozygous mice comprising two V kappasegments (Vκ1-39 and Vκ3-20) each containing three histidinesubstitutions (mice whose kappa locus is depicted in FIG. 33) andendogenous mouse heavy chains were harvested and homogenized in 1×PBS(Gibco) using glass slides. Cells were pelleted in a centrifuge (500×gfor 5 minutes), and red blood cells were lysed in ACK Lysis buffer(Gibco) for 3 minutes. Cells were washed with 1×PBS and filtered using a0.7 μm cell strainer. B-cells were isolated from spleen cells using MACSmagnetic positive selection for CD19 (Miltenyi Biotec). Total RNA wasisolated from pelleted B-cells using the RNeasy Plus kit (Qiagen).PolyA+ mRNA was isolated from total RNA using the Oligotex Direct mRNAmini kit (Qiagen).

Double-stranded cDNA was prepared from splenic B cell mRNA by 5′ RACEusing the SMARTer Pico cDNA Synthesis Kit (Clontech). The Clontechreverse transcriptase and dNTPs were substituted with Superscript II anddNTPs from Invitrogen. Immunoglobulin light chain repertoires wereamplified from the cDNA using primer specific for IgK constant regionand the SMARTer 5′ RACE primer (Table 14). PCR products were cleaned upusing a QIAquick PCR Purification Kit (Qiagen). A second round of PCRwas done using the same 5′ RACE primer and a nested 3′ primer specificfor the IgK constant region (Table 15). Second round PCR products werepurified using a SizeSelect E-gel system (Invitrogen). A third PCR wasperformed with primers that added 454 adapters and barcodes. Third roundPCR products were purified using Agencourt AMPure XP Beads. Purified PCRproducts were quantified by SYBR-qPCR using a KAPA LibraryQuantification Kit (KAPA Biosystems). Pooled libraries were subjected toemulsion PCR (emPCR) using the 454 GS Junior Titanium Series Lib-A emPCRKit (Roche Diagnostics) and bidirectional sequencing using Roche 454 GSJunior instrument according to the manufacturer's protocols.

TABLE 14 First Round PCR Primer NAME SEQUENCE (SEQ ID NO) 3′ mlgK outerAAGAAGCACACGACTGAGGCAC (SEQ ID NO: 129)

TABLE 15 Second Round PCR Primer NAME SEQUENCE (SEQ ID NO) 3′ mIgK innerGGAAGATGGATACAGTTGGTGC (SEQ ID NO: 130)

For bioinformatics analysis, the 454 sequence reads were sorted based onthe sample barcode perfect match and trimmed for quality. Sequences wereannotated based on alignment of rearranged Ig sequences to humangermline V and J segments database using local installation of igblast(NCBI, v2.2.25+). A sequence was marked as ambiguous and removed fromanalysis when multiple best hits with identical score were detected. Aset of perl scripts was developed to analyze results and store data inmysql database. CDR3 region of the kappa light chain was defined betweenconserved C codon and FGXG motif.

FIG. 34 represents alignments of amino acids sequence encoded by humangermline IGKV3-20 (FIG. 34A) or IGKV1-39 (FIG. 34B) sequence with aminoacid translations of exemplary Vκ sequences obtained from productivelyrearranged antibodies generated in mice comprising a histidine-modifiedDLC-5J (comprising a light chain variable locus comprising Vκ1-39 andVκ3-20 gene segments, each segment with three histidine modifications asdescribed above). The sequence reads showed that the majority ofproductively rearranged light chains retained at least one histidineintroduced into its germline CDR3. In some instances, in the majority ofall productively rearranged human light chains comprising Vκ3-20sequence that retain at least one histidine residue, all three histidinemodifications introduced into their germline CDR3 are retained (see FIG.34A). In some instances, in productively rearranged human light chainscomprising Vκ1-39 sequence that retain at least one histidine residue,about 50% of light chains retain all three histidines introduced intotheir germline CDR3 (see FIG. 34B top alignment), while about 50% oflight chains retain two out of three histidines introduced into theirgermline CDR3 (see FIG. 34B bottom alignment). In some instances,histidines at the last position of the V segment sequence may be lostdue to V-J rearrangement.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

What is claimed is:
 1. A genetically modified non-human animalcomprising in its germline an immunoglobulin light chain locuscomprising two unrearranged human Vκ_(κ) gene segments and one or moreunrearranged human Jκ gene segment operably linked to an immunoglobulinlight chain constant region sequence, wherein the two unrearranged humanVκ gene segments are human Vκ1-39 and Vκ3-20 gene segments eachcomprising one or more substitutions of a non-histidine codon with ahistidine codon, and wherein the human Vκ and Jκ gene segments arecapable of rearranging and encoding a human light chain variable domaincomprising one or more histidines at a position selected from the groupconsisting of 105, 106, 107, 108, 109, 111 (according to IGMTnumbering), and a combination thereof, wherein the one or morehistidines are derived from the one or more substitutions.
 2. The animalof claim 1, wherein the animal does not comprise an endogenous κ lightchain variable region gene segment that is capable of rearranging toform an immunoglobulin light chain.
 3. The animal of claim 1, whereinthe immunoglobulin light chain constant region sequence is a non-humanimmunoglobulin light chain constant region sequence.
 4. The animal ofclaim 3, wherein the non-human immunoglobulin light chain constantregion sequence is a mouse or a rat sequence.
 5. The animal of claim 3,wherein the non-human immunoglobulin light chain constant regionsequence is an endogenous immunoglobulin light chain constant regionsequence.
 6. The animal of claim 1, further comprising in its germlinean immunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region sequence comprising humanV_(H), D_(H), and J_(H) gene segments operably linked to animmunoglobulin heavy chain constant region sequence.
 7. The animal ofclaim 6, wherein the immunoglobulin heavy chain constant region sequenceis a non-human immunoglobulin heavy chain constant region sequence. 8.The animal of claim 7, wherein the non-human immunoglobulin heavy chainconstant region sequence is a mouse or a rat sequence.
 9. The animal ofclaim 7, wherein the non-human immunoglobulin heavy chain constantregion sequence is an endogenous non-human immunoglobulin heavy chainconstant region sequence.
 10. The animal of claim 1, wherein the twounrearranged human Vκ gene segments and the one or more unrearrangedhuman Jκ gene segment are present at the endogenous immunoglobulin lightchain locus.
 11. The animal of claim 1, wherein the immunoglobulin lightchain constant region is a Cκ region.
 12. The animal of claim 1, whereinthe animal is a rodent.
 13. The rodent of claim 12, wherein the rodentis a rat or a mouse.
 14. The rodent of claim 13, wherein the rodent is amouse.
 15. The animal of claim 1, wherein the animal comprises apopulation of B cells in response to an antigen of interest that isenriched for antibodies that exhibit a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.16. The animal of claim 15, wherein the enrichment in antibodies thatexhibit a decrease in t_(1/2) is at least about 2 fold.
 17. The animalof claim 1, wherein the animal comprises five human Jκ segments, and thefive human Jκ segments are human Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 segments.18. The animal of claim 1, wherein the immunoglobulin light chain locuscomprises no more than the human Vκ1-39 and Vκ3-20 gene segments and theone or more unrearranged human Jκ gene segment operably linked to animmunoglobulin light chain constant region sequence.
 19. The animal ofclaim 18, wherein either or both of the unrearranged human Vκ1-39 andVκ3-20 gene segments comprise a substitution of three or fournon-histidine codons with the histidine codons.
 20. The animal of claim19, wherein the substitution is of three non-histidine codons of thehuman Vκ1-39 gene segment, and the substitution is designed to expresshistidines at positions 106, 108, and 111 of the human Vκ1-39 genesegment.
 21. The animal of claim 19, wherein the substitution is of fournon-histidine codons of the human Vκ1-39 gene segment, and thesubstitution is designed to express histidines at positions 105, 106,108, and 111 of the human Vκ1-39 gene segment.
 22. The animal of claim19, wherein the substitution is of three non-histidine codons of thehuman Vκ3-20 gene segment, and the substitution is designed to expresshistidines at positions 105, 106, and 109 of the human Vκ3-20 genesegment.
 23. The animal of claim 19, wherein the substitution is of fournon-histidine codons of the human Vκ3-20 gene segment, and thesubstitution is designed to express histidines at positions 105, 106,107, and 109 of the human Vκ3-20 gene segment.
 24. The animal of claim1, wherein the animal expresses a population of antigen-specificantibodies in response to an antigen wherein all antibodies in thepopulation comprise: immunoglobulin light chain variable domains derivedfrom a rearrangement of the unrearranged human Vκ1-39 and Vκ3-20 genesegments and the one or more unrearranged human Jκ gene segment, andimmunoglobulin heavy chains comprising human heavy chain variabledomains derived from a repertoire of human heavy V, D, and J segments.25. A method of making a non-human animal that comprises a geneticallymodified immunoglobulin light chain locus in its germline, the methodcomprising: modifying a genome of the non-human animal to delete orrender non-functional endogenous immunoglobulin light chain Vκ and Jκgene segments in an immunoglobulin light chain locus, and placing in thegenome of the non-human animal an immunoglobulin light chain variableregion comprising two unrearranged human Vκ gene segments and at leastone unrearranged human Jκ gene segment, such that the immunoglobulinlight chain variable region sequence is operably linked to animmunoglobulin constant region sequence, wherein the two unrearrangedhuman Vκ gene segments are human Vκ1-39 and Vκ3-20 gene segments eachcomprising one or more substitutions of a one non-histidine codon with ahistidine codon, and wherein the unrearranged human Vκ and Jκ genesegments are capable of rearranging and encoding a human light chainvariable domain comprising one or more histidines at a position selectedfrom the group consisting of 105, 106, 107, 108, 109, 111 (according toIGMT numbering) and a combination thereof, wherein the one or morehistidines are derived from the one or more substitutions.
 26. Themethod of claim 25, wherein the immunoglobulin light chain variableregion is at the endogenous non-human immunoglobulin light chain locus.27. The method of claim 25, wherein the animal is a rodent.
 28. Themethod of claim 27, wherein the rodent is a mouse or a rat.
 29. A methodof generating an antibody that exhibits pH-dependent binding to anantigen of interest comprising: immunizing the animal of claim 1 with anantigen of interest.
 30. The method of claim 29, further comprisingselecting an antibody that binds to the antigen of interest with adesired affinity at a neutral pH while displaying reduced binding to theantigen of interest at an acidic pH.